Tesis Doctoral Por Estefanía Ledesma Santiso Abril, 2015

Departamento de Ingeniería Química y Tecnología del Medio Ambiente Programa de Doctorado en Biotecnología Alimentaria

Benzo(a)pyrene control and transport processes in smoked meat products

Tesis doctoral por Estefanía Ledesma Santiso Abril, 2015

Agradecimientos

AGRADECIMIENTOS•

Que mejor manera de comenzar esta memoria que haciendo mención a todas las personas que han participado en ella, mi ilusión. Y digo bien, a todas las personas.

Por favor, permítanme que les pida que cierren de nuevo el libro y miren la portada. ¿Ven las primeras palabras?, Universidad de Oviedo, Departamento de Ingeniería Química y Tecnología del Medio Ambiente, Doctorado en Biotecnología Alimentaria. Estas palabras dicen muchas cosas, dicen que esta tesis doctoral se ha realizado en este centro, respaldado por todas las personas que han participado en mi formación hasta el día de hoy.

Entre ellas quiero expresar mi primer agradecimiento, al Dr. Mario Díaz Fernández y al Dr. Manuel Rendueles de la Vega, por darme esta oportunidad, y por sus conocimientos, experiencia, dirección, orientación y apoyo. Agradezco su vitalidad, y dedicación intensa en su trabajo, que afortunadamente para mí, ha incluido dirigir esta tesis. Particularmente agradezco su habilidad para ordenar mis ideas, darles sentido, y ayudarme a ejecutarlas y expresarlas. Agradezco sus tantas horas extra (si, fines de semana y vacaciones) que me han dedicado, manteniendo la fe en que culminaría mi estudios, pese a la dificultad de combinarlos con el trabajo. Me gustaría agradecer algo concreto de cada uno de ellos. En particular, la capacidad “catalizadora” de Mario, rápidamente ordenando, acelerando e impulsando las ideas en la correcta dirección, e intentando sacar el mayor provecho del trabajo realizado, y de las personas que lo hacen, y de Manolo, además, por la paciencia en el día a día, su cercanía, su rapidez en responder a toda pregunta por redundante que sea, por las explicaciones y correcciones (especialmente por la simpatía en expresarlas para acogerlas mejor), y sobre todo por sus palabras de apoyo en los momentos difíciles, por el “saldrá” .

Deseo expresar mi agradecimiento al Programa de Doctorado en Biotecnología Alimentaria y a todo el personal de la Universidad de Oviedo. En particular, agradezco el trabajo, toda la información y apoyo de la Dra. Adriana Laca, Coordinadora del Programa de Doctorado, y todos los profesores que han intervenido en mi formación, en concreto en el Máster en Biotecnología Alimentaria. Especialmente, por la cercanía antes y durante el inicio de la tesis, les ofrezco un sincero y sentido agradecimiento a los doctores Ricardo Álvarez y Francisco Riera. Agradecimientos

Me gustaría expresar mi gratitud a todos mis compañeros y personal científico técnico de la Universidad de Oviedo. El laboratorio no sería lo mismo sin la Dra. Amanda Laca, llenándolo de alegría y actividad, con una mente tan despierta, siendo a la par ordenada y curiosa en su trabajo, cualidades que me han animado tanto en la lucha contra la impaciencia y desesperación con algunos equipos y experimentos. Al Dr. Carlos Álvarez, por ofrecerme su ayuda siempre que lo necesité, espero que el futuro te devuelva todo lo bueno que te mereces. Al Dr. Saúl Alonso, por su ayuda, consejos y perseverancia en su trabajo, un claro ejemplo de compañerismo y profesionalidad. A la Dra. Paula Oulego, por ser tan trabajadora, alegre y cordial, en particular por compartir conmigo la línea del Nitrógeno (que tantas veces me ha ayudado utilizar el Dr. Sergio Collado, muchas gracias Sergio). A los doctores Ainhoa Blanco, Emilio Muñoz y Pepe Cervero, por sus conocimientos sobre cromatografía de gases/espectrometría de masas (GC/MS), fundamentales para el desarrollo de esta tesis. A Bea, por enseñarme a utilizar el sistema de extracción en fase sólida (SPE). A todos los compañeros y/o doctores del departamento que han hecho agradable la estancia allí, especialmente a Silvia, Pilar, Ana, Ismael, Daniel, Violeta, Ayoa, Noel, Miguel, María, Leticia, Celeste, Rosana, Janire, Leticia, Estefanía y Federico.

Quisiera expresar mi agradecimiento al equipo de los Servicios Científico-Técnicos de la Universidad de Oviedo (SCTs), y colaboradores. En concreto, a los doctores Marta Alonso, Ángel Martínez (Unidad de Microscopía Fotónica y Proceso de Imágenes), y Alfredo Quintana (Unidad de Microscopia Electrónica). Gracias por vuestra gran ayuda, siempre agradable trato, colaboración, y aportaciones a este trabajo, en especial por ayudarme a ver más de lo que podía imaginar. También, me gustaría dar un gran agradecimiento al Dr. Giuseppe Centineo, Julio Fernández, Rubén García y Julio Rodríguez, trabajadores de Agilent Technologies e ISC-Science, por ayudarme a entender y utilizar el GC/MS. En particular la ayuda de Julio y Giuseppe ha sido definitivamente clave para conseguir arrancar y avanzar. Me gustaría mucho dar las gracias a Paul Barnes, que casi en la sombra, como un duendecillo en el ordenador, ha revisado todos los artículos escritos en inglés en este trabajo. Muchas gracias Paul.

Así mismo, les doy las gracias a todos los profesores que desde la infancia han animado mi carrera en ciencias, y a todos los compañeros y amigos que me han acompañado, especialmente, claro que si, a Nausika Querejeta Montes. Agradecimientos

Quisiera expresar especialmente mi gratitud a todas las entidades y personas que impulsan y hacen posible la ciencia en España, como FICYT, IDEPA, CEEI, CDTI, y MINECO. Así mismo, agradezco la labor realizada por las entidades y revisores externos de las publicaciones que conforman esta memoria.

Por otro lado, me gustaría agradecer tantas cosas a la empresa que me ha permitido hacer este trabajo, Embutidos El Hórreo S.L, que he visto convertirse en El Hórreo Healthy Food S.L. Me gustaría agradecer a todos ellos, Joaquín, Carmen (en paz descansen), Sese, Jaki y Carmen, y al resto de compañeros, Lorena, María Elena, Joaquín, Susana, Eva y Elena, su alegría y ánimo en el día a día. Son un claro ejemplo de esfuerzo y lucha para avanzar, y alcanzar nuevos horizontes, a la par de humanidad, respeto a la tradición, y responsabilidad para salvaguardar a un colectivo. Les agradezco su confianza y apoyo para llevar a cabo mis experimentos y los proyectos que pasan por mi cabeza. Hemos pasado por momentos alegres, tristes, victorias y derrotas, pero siempre se ha valorado el esfuerzo fuese cual fuese el resultado.

Volvamos una vez más a la portada del libro. ¿Ven algún nombre más? ¿Estefanía Ledesma Santiso? Este no es más que el resultado de Ledesma & Santiso (1985) que pusieron de título a su trabajo “Estefanía”, junto a todas las personas que estos apellidos aúnan y me han formado hasta el día de hoy, aña damos pues “et al. (1985 -2015)”. Todos ellos son autores de estas líneas, pues el resultado de mi trabajo es el suyo. Darles las gracias de cualquier cosa en particular es poco, pues les doy las gracias por todo, por la vida y llenarla de sentido y felicidad. En particular, c on cariño les doy gracias a mis padres por ser un ejemplo desde “sus cumbres, hasta mis picos” , a mis hermanas Maleny y Carol, y a mi “cuñadohermano” Santi, y por supuesto a la mejor animadora del mundo, Alex. Mejor sería que les pidiera disculpas, por el tiempo y cordura dedicado a este empeño, dejando escapar tantos momentos con ellos. En mi familia, agradecimientos y disculpas, incluyo a “los Riazas ” y los “Benito”, que me han acogido como uno más. Entre todos ellos, guardo para siempre mi agradecimiento más especial, últimas palabras y pensamientos para mi “principal investigador”, el Dr. Juan Riaza, por ser mi alegría, esperanza, apoyo, y aliento en cada instante.

Finalmente le doy gracias a Dios, por haberme hecho encontrar con todos ellos.

A todas las personas que dedican su vida en mejorar

el bienestar y la salud humana.

Indice

••ÍNDICE•

RESUMEN V ABSTRACT VII

1. INTRODUCCIÓN 3 1.1. Introducción 3 1.2. Objetivos 9 1.3. Estructura de la memoria 11

2. CONSIDERACIONES TEÓRICAS 17 2.1. Smoked Food 19

3. MATERIALES Y MÉTODOS 85 3.1. Obtención de muestras 85 3.1.1. Tripas 85 3.1.2. Chorizo 86 3.1.3. Aceite 91 3.2. Determinación de benzo(a)pireno 93 3.2.1. Reactivos y patrones 93 3.2.2. Pretratamiento de muestras de chorizo 94 3.2.3. Pretratamiento de muestras de aceite 100 3.2.4. Pretratamiento de las tripas 100 3.2.5. Determinación de BaP mediante cromatografía de gases/ espectrometría 100 de masas (GS/MS) 3.3. Caracterización física del efecto de las tripas en el chorizo 103 3.3.1. Caracterización de las tripas 103 3.3.2. Chorizos embutidos en tripas 106 3.4. Análisis estadísticos 110

Indice

4. RESULTADOS 113 4.1. Determinación de B(a)P en chorizos ahumados del Principado de Asturias 113 4.2. Mecanismo de penetración de B(a)P en chorizo: Influencia del tiempo de 123 ahumado y la profundidad 4.3. Influencia del tipo de tripa en la penetración de B(a)P en chorizo ahumado 137 4.4. Influencia del tipo de tripa en las propiedades del chorizo durante el ahumado: 151 textura, color y características ópticas

5. DISCUSIÓN GENERA L 327 5.1. Discusión de resultados 337

6. CONCLUSIONES 247

7. BIBLIOGRAFÍA 253

8. ANEXOS 269 8.1 Difusión de la tesis doctoral 269 8.1.1. Artículos científicos 269 8.1.2. Comunicaciones a congresos 270 8.2 Informe del factor de impacto de las publicaciones presentadas 271 8.3 Nomenclatura 272 8.3.1 Acrónimos 272 8.3.2 Abreviaturas 273 8.3.3 Símbolos 274 8.3.4 Símbolos con letras griegas 275

Indice

••Lista•de•figuras•

Figura 1.1 Elaboración propia con datos de “Encuesta Industrial de Empresas 3 2013” (INE, 2014).

Figura 1.2 Chorizo. 4

Figura 1.3 Benzo(a)pireno. 6

Figura 1.4 Chorizo ahumando. 7

Figura 3.1 Picado (a) y amasado (b) de los ingredientes para la fabricación de 86 muestras de chorizo.

Figura 3.2 Selección y remojado de la tripa. 87

Figura 3.3 Proceso de embutido de la masa en la tripa. 87

Figura 3.4 Colocación de chorizo en estantería para llevar a la cámara de 88 ahumado.

Figura 3.5 Cámara de ahumado directo industrial (tradicional) de El Hórreo 89 Healthy Food S.L.

Figura 3.6 Diagrama de flujo del proceso de fabricación de muestras de chorizo . 90

Figura 3.7 Selección y medida (a), remojo (b) y llenado (c) de las tripas. 91

Figura 3.8 Sistema de aceite en distintos tipo s de tripa, colgado en la estantería. 92

Figura 3.9 Transporte de sistemas de aceite en distintos tipos de tripa. 92

Figura 3.10 Preparación de sistema de aceite en tripa natural y colágeno, y toma 93 de muestras.

Figura 3.11 Pretratamiento. 94

Figura 3.12 Selección y cuarteamiento de muestras . 95

Figura 3.13 Equipo de liofilización. 96

Figura 3.14 Filtrado. 97

III

Indice

Figura 3.15 Equipo SPE. 97

Figura 3.16 Proceso de extracción en fase sólida, de BaP en productos cárnicos 98 ahumados.

Figura 3.17 Vial. 99

Figura 3.18 Cromatógrafo de gases (GC) 6890/ espectrómetro de masas (MS) 103 5975, Agilent Technologies.

Figura 3.19 Procedimiento de secado (a) y transporte (b) de membranas (tripas). 104

Figura 3.20 Porosímetro Micromeritics Autopore IV. 105

Figura 3.21 Microscopio electrónico de barrido MEB JEOL-6610LV SCTs UO. 105

Figura 3.22 Microscopio de Fluorescencia Óptica Leica M205 FA. SCTs UO. 106

Figura 3.23 Colorímetro. 107

Figura 3.24 Texturómetro. 108

Figura 3 .25 Pocillos. 109

Figura 3.26 Dispositivo para determinación de humedad: desecador, arena, 109 pocillos y horno.

Figura 8.1 Revistas científicas de difusión de la presente tesis doctoral. 269

Figura 8.2 ICCE. 270

Figura 8.3 INDC 270

Figura 8.4 Present ación de poster INDC. Brno, República Checa, 28- 31 de 270 agosto, 2011.

Resumen

••RESUMEN•

El incremento del consumo de carne y productos cárnicos en el mundo, así como su proyección en los países en desarrollo y desarrollados, ha estimulado la investigación de su impacto en la salud humana. Los productos cárnicos son componentes importantes de una dieta saludable y balanceada, destacando su elevado contenido proteico. El ahumado es una de las técnicas más antiguas para prolongar la vida útil de los alimentos, siendo utilizado con este objetivo en algunos países en desarrollo. Además confiere buenas propiedades organolépticas a los productos, por lo que todavía es aplicado en el 60% de los productos, en algunos países desarrollados. Las investigaciones se han focalizado en el aporte de sustancias cancerígenas a los productos cárnicos durante su procesado, destacando la contaminación por hidrocarburos aromáticos policíclicos (HAP) durante el ahumado directo. En este contexto, el Codex Alimentarius (CAC/RCP 68/2009) ofrece una guía del proceso de ahumado, y el contenido máximo de benzo(a)pireno (BaP) (un marcador de la presencia y efecto de HAP en alimentos), se fijó en 5 •g/kg, y se redujo a 2 •g/kg desde el 1/9/2014, por los reglamentos europeos No.1881/2006 y 835/2011, respectivamente. En esta tesis doctoral, se aborda el estudio de los procesos de transporte y contaminación por BaP, durante el ahumado directo de uno de los productos cárnicos españoles más conocidos, el chorizo, elaborado en una zona no estudiada antes. Además, se investigan las posibles causas para su prevención.

Para ello, se ha desarrollado un método analítico basado en la combinación de las técnicas de pretratamiento de muestra, sonicación y extracción en fase sólida (SPE), y la determinación mediante cromatografía de gases/espectrometría de masas (GC/MS). Este método ha sido aplicado, en primer lugar, en chorizos ahumados elaborados por 16 empresas del Principado de Asturias, confirmando que 5 sobrepasan el límite reglamentario de BaP. La falta de correlación con su humedad, revela la necesidad de optimizar el ahumado directo como método de secado.

En segundo lugar, se determinó la influencia del tiempo de ahumado directo, variable recomendada por el Codex Alimentarius, en la contaminación de chorizo por BaP, y se estudio por primera vez, la penetración de BaP en distintas profundidades del chorizo ahumado. Se demostró que el incremento del tiempo de ahumado, produce efectos contrarios entre el contenido de BaP y la humedad del chorizo. El contenido de humedad decrece, y el contenido de

Resumen

BaP aumenta, y finalmente se estabiliza entre los 5 y 7 días de ahumado, con un valor por debajo del límite legal europeo. El contenido de BaP del chorizo disminuye desde la tripa, donde supera 10 veces el límite legal, hasta el interior del chorizo.

Posteriormente, en el tercer trabajo, se estudió la influencia del uso de distintos tipos de tripa, natural y sintética (de colágeno), en la penetración de BaP en chorizo ahumado, encontrando grandes diferencias. Para explicarlas, se diseñaron sistemas novedosos, que permiten la caracterización de las propiedades físicas de las tripas, mediante porosimetría, y se estudió la evolución de estos chorizos durante el ahumado, mediante imágenes tomadas con estereomicroscopio de fluorescencia óptica, y microscopio electrónico de barrido (SEM). Esta tesis evidencia, y explica por primera vez, la capacidad de las tripas sintéticas para prevenir la penetración de BaP dentro de chorizo ahumado. Los resultados encontrados permitieron proponer, por primera vez, un mecanismo que explica la contaminación por BaP de los productos cárnicos embutidos en tripa natural, durante el ahumado directo.

Finalmente, en un cuarto estudio, se compararon las propiedades, textura, color y humedad, de los chorizos embutidos en los 2 tipos de tripa, durante el ahumado. Esta tesis demuestra que ambos tipos, confieren buenas propiedades al chorizo, pero la tripa de colágeno permite reducir el tiempo de procesado, garantiza la estandarización, y previene la contaminación por BaP en el interior del producto, si es ahumado. Esta tesis doctoral recoge un análisis detallado bibliográfico de los alimentos ahumados, el proceso tecnológico de ahumado y los beneficios y desventajas que confiere, destacando la contaminación por HAP. Finalmente, se describen e identifican las variables del proceso más importantes a controlar para prevenir la contaminación de los productos cárnicos por HAP durante el ahumado.

La presente tesis doctoral ofrece datos científicos relevantes, basados en el control de variables del ahumado recomendadas por el Codex Alimentarius, que permiten a los fabricantes minimizar el contenido de BaP en chorizo ahumado a modo directo, con el objetivo de fabricar productos cárnicos más saludables, y respetar el nuevo límite de BaP fijado por la normativa europea.

Abstract

••ABSTRACT•

The increase of global meat and meat products consumption, and its projection in developing and developed countries has stimulated research concerning its impact to human health. Meat products are an important component of a healthy and well balanced diet, mainly because of its high level protein content. The smoking process is one of the oldest techniques for prolonging food shelf life. It is applied with this aim in some developing countries. Moreover it is still applied to 60% of the meat products in some developed countries because of the special organoleptic profile that it confers. Research is focused in the addition of carcinogenic compounds to meat products during processing, standing out the contamination by polycyclic aromatic hydrocarbons (PAH) during direct smoking. Within this context, Codex Alimentarius (CAC/RCP 68/2009) provides guideline of smoking process, and the maximum permissible content of benzo(a)pyrene (BaP) (a marker for the occurrence and effect of PAH in foods) in smoked meat products was fixed in 5 •g/kg , and reduced to 2 •g/kg from 1/9/2014 on, by European Regulations No. 1881/2006 and No. 835/2011, respectively. This PhD dissertation is focused in the research of the transport processes and contamination by BaP during direct smoking process of a well known Spanish meat product called chorizo, manufactured in an as-yet unstudied region. Moreover the possible causes are studied, in order to prevent it.

An analytical method was developed for this purpose, consisting of PAH extraction assisted by sonication, followed by solid-phase extraction (SPE) sample clean-up, and analytical determination using Gas Chromatography/Mass Spectrometry (GC/MS). Firstly, this method was applied to smoked chorizos made by 16 different producers from the Principality of Asturias. It was found that 5 of the samples exceeded the BaP legal limit. As not correlation between moisture and BaP content was found, the necessity of the optimization of direct smoking process was demonstrated.

Secondly, the influence of direct smoking time, variable advised by Codex Alimentarius, in the BaP contamination of chorizo was determined, and the penetration of BaP in different depths in the smoked chorizo was studied for the first time. It was proved that an increase in smoking time produces opposite effects on the moisture and the BaP content of chorizo. While the moisture content decreases, the BaP content increases finally becoming stabilized after 5 and 7 days of smoking. Then, a concentration below the legal limit was found. The BaP content

VII

Abstract

decreases from the casing, where a value 10 times over the legal limit was found, towards the inside of the chorizo.

Afterwards, in the third work, the influence of casing types, natural and synthetic (collagen), in the BaP penetration in smoked chorizo was studied, finding large differences. In order to find the reasons, new systems were developed, allowing the physical characterization of the casings by porosimetry, and the evolution of chorizos during smoking was studied by means of Fluorescence Stereo Microscope and scanning electron microscopy (SEM) images. The capacity of synthetic casings to prevent the BaP penetration into smoked chorizo was shown, and explained by first time. According with the findings, a new mechanism explaining the BaP contamination of chorizo stuffed in natural casing during direct smoking was proposed by first time.

Finally, in the fourth work, the texture, color and humidity properties of chorizos stuffed in both types of casing during smoking time were compared. This thesis proves that both types of casings give good properties to chorizo, but synthetic casing allows the reduction of processing time, enables the standardization, and prevents the BaP contamination into the chorizo, in case of smoking application. This thesis includes detailed bibliographic reviews about smoked food, the smoking process and the benefits and disadvantages that it confers, standing out the contamination of food by PAH. Finally, the most important variables affecting the smoking process are defined and identified, in order to prevent the PAH contamination of meat products during smoking process.

The present dissertation provides relevant scientific dates, based on the control of smoking process variables advised by Codex Alimentarius, allowing producers to minimize the BaP content of direct smoked chorizo, with the aim of produce healthier meat products, and respect the new BaP legal limit fixed by the European regulation.

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•••••••••••••••••••••••••••••••••••••••••••••••••••Introducción••• • • • • • • • Introducción

1. INTRODUCCIÓN

1.1 INTRODUCCIÓN

La carne y los productos cárnicos han tenido un rol crucial en la evolución humana y son componentes importantes de una dieta saludable y balanceada (De Castro Cardoso Pereira & Dos Reis Baltazar Vicente, 2013). Desde el punto de vista nutricional, su importancia deriva de su elevado contenido en proteína, siendo una alimento que contiene aminoácidos esenciales, así como minerales, vitaminas, micronutrientes como el hierro, selenio, zinc y vitamina B12 (De Castro Cardoso Pereira & Dos Reis Baltazar Vicente, 2013; FAO, 2014a; OMS/FAO, 2003; USDA/HHS, 2010). Las vísceras como el hígado son además fuentes cruciales de vitamina A y ácido fólico (Biesalski, 2005).

Dada su importancia para la evolución humana, estos productos han adquirido un elevado interés económico, siendo actualmente los alimentos pecuarios de mayor valor. Tal y como refleja la figura 1, en España, la industria cárnica, con 22.167 millones de euros, lidera el sector de la Alimentación. Representa el 25,4% de éste, primera actividad económica del sector industrial, y el 3,94% del total de la industria (INE, 2014).

Figura 1.1. Elaboración propia con datos de “Encuesta Industrial de Empresas 2013” (INE, 2014).

Capítulo 1

Sin embargo, la industria cárnica se caracteriza por su elevada atomización, estando formada por un elevado número de empresas de pequeño tamaño. En España hay 4.036 empresas cárnicas (14% del número total de empresas alimentarias), de las cuales el 70,8% tiene menos de 9 asalariados y el 23,7% entre 10 y 49, generando en total el 22,90% de empleo de la industria alimentaria. Entre todas ellas, tan solo 10 empresas generan el 43% de las ventas totales del sector cárnico (MAGRAMA, 2014a). Esto hace que la gran mayoría de empresas cárnicas no puedan actualizar sus instalaciones con modernos y costosos sistemas de producción. Se proyecta que el consumo de carne en el mundo se duplique en el año 2050 (FAO, 2014a). Con un consumo per cápita medio de 15,3 kg/año, los productos cárnicos de cerdo serán en 2015 los más consumidos a nivel mundial (FAO, 2014b), habiéndose comercializado 110.270 miles de toneladas mundiales en 2011 (FAO, 2014c). España es la cuarta potencia productora de ellos (después de China, EEUU, y Alemania) (ANICE, 2013; MAGRAMA, 2013).

Uno de los productos cárnicos embutidos españoles, elaborados entre otras materias primas con carne de cerdo, más importante y conocido es el chorizo. En el año 2013, se consumieron en España 53.011,54 toneladas de chorizos, generando 435,81 millones de euros para las empresas productoras (MAGRAMA, 2014b). Existen varias tipologías de chorizos tradicionales y denominaciones desarrolladas por el uso, reguladas recientemente por la nueva norma de calidad de Figura 1.2. Chorizo. productos cárnicos. Entre ellas se encuentran el chorizo criollo, chorizo cular, chorizo de cebolla, chorizo de entraña, chorizo de Pamplona, chorizo de Teror, chorizo palmero, chorizo de perro, chorizo rondeño y chorizo sabadiego (BOE, 2014). Además existen 2 chorizos con Indicaciones Geográficas Protegidas (IGP), el chorizo de Cantimpalos y el chorizo Riojano (MAGRAMA, 2014c). El choriz o asturiano está protegido y diferenciado bajo “marca colectiva” gracias al esfuerzo de entidades como el Centro Tecnológico Agroalimentario “Asociación de Industrias Cárnicas del Principado de Asturias” (ASINCAR), en colaboración con la Consejería de Agroganadería y Recursos Autóctonos (ASINCAR, 2015a). En el Principado de Asturias hay unas 73 empresas cárnicas (ASINCAR, 2015b), y se consumen 2.283,30 toneladas de chorizos al año (MAGRAMA, 2014b). Los chorizos elaborados en el Principado de Asturias, además de contener magro y panceta de cerdo, magro de vacuno ocasionalmente, sal, pimentón, ajo y otras especias, se caracterizan principalmente por sus cualidades organolépticas derivadas

Introducción

de la exposición al ahumado. El ahumado es aplicado al modo tradicional en Asturias y otras regiones y países. Entre ellos destacan las zonas en desarrollo como África y Asia, donde la cadena de refrigeración no ha sido todavía establecida (FAO-Thiaroye, 2015; Ogbadu, 2014; Vaz- Velho, 2003) . La técnica de ahumado optimizada ha sido implantada en algunos países desarrollados. El ahumado es muy aplicado en los productos cárnicos a nivel mundial. Por ejemplo, en Alemania se ahúman el 60% de los productos cárnicos (Frede, 2006).

El ahumado es una de las técnicas más antiguas de conservación de los alimentos, siendo probablemente los productos cárnicos los primeros alimentos ahumados por el hombre ( Šimko, 2002, 2009). El humo está compuesto por numerosas sustancias químicas, habiéndose identificado más de 1100 compuestos (Wilms, 2000). Estos compuestos pueden clasificarse en muchos grupos, pero ejercen principalmente 2 efectos en productos cárnicos, efectos deseados y no deseados. Entre los efectos deseados destacan la prolongación de la vida útil y mejora de las propiedades organolépticas de los alimentos. Entre los compuestos que ayudan a prolongar la vida útil se encuentran algunos ácidos orgánicos y carbonilos (Milly, Toledo, & Chen, 2008; Montazeri, Himelbloom, Oliveira, Leigh, & Crapo, 2013), algunos compuestos fenólicos, como el fenol o el isoeugenol (Suñen, 1998; Young & Foegeding, 1993) que ejercen actividad antimicrobiológica frente a Listeria monocytogenes, otros compuestos bactericidas frente a Salmonella Typhimurium (Kim, Kang, Park, Nam, & Friedman, 2012), Escherichia coli (Van Loo, Babu, Crandall, & Ricke, 2012), Staphylococcus aureus y enterotoxinas estafilocócicas (Taormina & Bartholomew, 2005), y otros muchos compuestos con actividad biocida y fungicida (Möhler, 1978). El efecto de estas sustancias junto con el descenso de la actividad de agua de los alimentos durante el secado producen el efecto preservativo conocido del ahumado (Vaz-Velho, 2003). Además el ahumado mejora las propiedades organolépticas, color, textura y flavor de los alimentos (Adhikari, Heymann, & Huff, 2003; Ahmad, 2003; Birkeland, Bencze Rørå, Skåra, & Bjerkeng, 2004; Bozkurt & Bayram, 2006; Cardinal et al., 2001; Kim et al., 2014; Kostyra & Bary!ko -Pikielna, 2006; Möhler, 1978; Pöhlmann, Hitzel, Schwägele, Speer, & Jira, 2013a; Prändl, Fischer, Schmidhofer, & Sinell, 1994 ; Šimko, 2002, 2009; Vaz-Velho, 2003; Woods, 2003). Entre las sustancias que provocan el flavor y olor típico de los alimentos ahumados destacan los compuestos fenólicos, fenol, p-cresol y o-cresol, y los carbonilos, cicloten y 3- methylociclopenteno (Kostyra & Bary!ko -Pikielna, 2006).

Capítulo 1

Por otro lado el ahumado produce efectos no deseados en los alimentos, entre los que destaca la contaminación por sustancias tóxicas y cancerígenas, como los hidrocarburos aromáticos policíclicos (HAP) (CCA, 2009), las n-nitrosaminas (Herrmann, Duedahl-Olesen, & Granby, 2015; Yurchenko, & Mölder, 2007), las aminas aromáticas heterocíclicas (Naccari et al., 2009; Simon, De la Calle, Palme, Meier, & Anklam, 2005), y los •-carbonilos (Papavergou & Herraiz, 2003; Sen, Seaman, Lau, Weber & Lewis, 1995). Entre todas ellas, las investigaciones se han focalizado en los HAP, debido a su elevada actividad cancerígena (FAO/OMS, 2006; SCF, 2002). La principal fuente de contaminación por HAP para el ser humano es la dieta (Falcó, Domingo, Llobet, Teixidó, Casas, & Müller, 2003; Ibáñez et al., 2005; Lodovici, Dolara, Casalini, Ciappellano, & Testolin, 1995; Phillips, 1999), contribuyendo al 70% de exposición en los no fumadores (Gilbert, 1994; McGrath, Wooten, Geoffrey Chan, & Hajaligol, 2007). Se han reportado niveles elevados de HAP en numerosos alimentos ahumados y también no ahumados, probablemente debido a contaminación medioambiental (CCA, 2009; Rodríguez-Acuña, Pérez- Camino, Cert, & Moreda, 2008). De entre todos los alimentos, desde una perspectiva global, los mayores contribuyentes de HAP son los cereales y los aceites y grasas vegetales (CCA, 2009), debido al elevado nivel de consumo a nivel mundial. Sin embargo, algunas investigaciones indican que los mayores contribuyentes de HAP en la dieta son los productos cárnicos ahumados (Martorell et al., 2010), ya que en ellos se encontraron los niveles más elevados de HAP (Gomaa, Gray, Rabie, López-Bote, & Booren, 1993; Karl & Leinemann, 1996; Larsson, Pyysalo, & Sauri, 1988; Martorell et al., 2010).

Así mismo, se ha asociado el consumo de carne y productos cárnicos con un aumento en el riesgo de contraer ciertos tipos de enfermedades crónicas, destacando el cáncer colorectal (CCR) (Alexander et al., 2010; Alexander et al., 2011; Aune et al., 2013; Biesalski, 2005; Corpet, 2011; Demeyer et al., 2008; Ferguson, 2010; McNeill & Van Elswyk, 2012; OMS/FAO, 2003; WCRF/AICR, 2007; Wyness et al., 2011), primera causa de cáncer en la población Europea (con acerca de 28,2 casos por 100.000 habitantes al año), segunda en mujeres a nivel mundial (614,000 casos, 9,2% del total) y tercera en hombres a nivel mundial (746,000 cases, 10,0% del total) (IARC, 2012). Esta relación ha sido rechazada recientemente (McAfee et al., 2010), ya que existe alguna inconsistencia entre los datos observacionales y experimentales entre carne y cáncer (Dragsted et al., 2014). Se concluyó que se precisa una mayor revisión (Kim et al., 2013). Por todo ello, es necesario establecer medidas para la prevención del aumento de la actividad

Introducción

carcinogénica de los productos cárnicos derivada de su procesado, en particular del ahumado directo.

Para proteger a los consumidores en la ingesta de HAP, el contenido máximo permitido de estas sustancias en algunos alimentos fue regulado por el reglamento europeo N o 1881/2006, estableciendo al benzo(a)pireno (BaP) como el marcador de la presencia y efecto de los HAP en los alimentos, Figura 1.3. Benzo(a)pireno. incluidos la carne ahumada y los productos cárnicos ahumados. Estudios previos indican que el BaP es el HAP más peligroso (Šimko, 2002). Recientemente (Septiembre, 2014) este contenido máximo ha sido reducido hasta 2 !g/kg por el reglamento (EU) N o 835/2011, añadiendo también como control la suma de 4 sustancias (HAP4) (BaP, benzo(a)antraceno, benzo(b)fluoranteno y criseno), manteniendo siempre un control separado para el BaP, para poder comparar con los datos previos y futuros. Un estudio reciente (Lorenzo et al., 2011) realizado para comprobar su idoneidad, concluye que el BaP es el marcador idóneo de la presencia de los 16 (reglamentación europea) o 7 (US EPA) HAP carcinogénicos reconocidos por la reglamentación, en concreto en chorizo gallego, muy similar al asturiano.

La contaminación por HAP de los productos cárnicos durante el ahumado puede ser controlada, para mantener los buenos efectos y prevenir los malos. En particular, la Comisión del Códex Alimentarius (CCA, 2009) proporciona las 10 variables que deben ser controladas para minimizar y prevenir la contaminación de los productos cárnicos por los carcinogénicos HAP durante el ahumado. El proceso tecnológico de ahumado es muy antiguo y ha sido sustituido en los países desarrollados por otras técnicas de conservación y procesado que permiten prolongar la vida útil (Zhou, Xu, & Liu, 2010), y prevenir la contaminación por HAP de los productos cárnicos, como por ejemplo sistemas de ahumado indirecto (Pöhlmann et al., 2013b). Sin embargo, la implantación de estos procesos es muy costosa e imposible para las comunidades de los países en desarrollo (FAO-Thiaroye, 2015; Ogbadu, 2014; Vaz-Velho, 2003), y las pequeñas empresas de la atomizada industria cárnica de los países desarrollados.

Capítulo 1

La prevención de la contaminación por HAP de chorizo ahumado a modo directo es un tema de elevada importancia para garantizar la seguridad alimentaria e inocuidad de estos alimentos, y en definitiva proteger la salud humana.

Figura 1.4. Chorizo ahumando.

Introducción

1.2 OBJETIVOS

Así pues, a la vista de la necesidad de profundizar en los problemas de seguridad alimentaria derivados del consumo de productos cárnicos ahumados, se ha planteado como Objetivo General (O.G) de esta tesis doctoral definir y determinar la contaminación por benzo(a)pireno (BaP) de chorizos ahumados del Principado de Asturias y sus mecanismos de transporte, proponiendo métodos para su prevención.

Para ello se plantean los siguientes objetivos específicos (O.E.) :

O.E.1 Revisar los estudios que la comunidad científica ha desarrollado en relación a la contaminación de productos cárnicos ahumados por hidrocarburos aromáticos policíclicos (HAP), y analizar los métodos propuestos para su prevención.

O.E.2 Poner a punto un método analítico para la determinación de BaP en chorizo ahumado.

O.E.3 Determinar la presencia de BaP en chorizos ahumados, fabricados por diferentes empresas del Principado de Asturias, y analizar su contenido en humedad para evaluar el proceso de secado.

O.E.4 Evaluar la influencia de los siguientes parámetros del proceso de ahumado directo en la presencia de BaP en chorizo del Principado de Asturias:

a) El tiempo de ahumado.

b) El tipo de tripa: natural y sintética.

O.E.5 Estudiar la penetración de BaP en distintas profundidades de chorizo ahumado.

O.E.6 Caracterizar las diferencias físicas entre las tripas naturales y sintéticas y su influencia en la penetración de BaP en chorizo ahumado.

O.E.7 Proponer un mecanismo que explique la contaminación de chorizo por BaP durante el ahumado.

O.E.8 Caracterizar la influencia de las tripas natural y sintética en las cualidades organolépticas, color y textura, de chorizo ahumado.

Introducción

1.3 ESTRUCTURA DE LA MEMORIA

La estructura de la presente memoria de tesis doctoral se presenta como convenio de publicaciones llevadas a cabo en la línea de investigación “contaminación por benzo(a)pireno de chorizos ahumados del Principado de Asturias y las posibles causas para su prevención ”. Cada una de las publicaciones tiene una estructura común, de acuerdo al esquema tradicional, constando de un resumen, una introducción, la descripción de los materiales y metodología empleados, la presentación y discusión de los resultados obtenidos, las conclusiones, los agradecimientos y las referencias utilizadas. Tres de los artículos presentados han sido ya publicados en revisas científicas incluidas en el Science Citation Index, incluyendo además otras que han sido enviadas. La estructura de la memoria presentada, está formada por 8 capítulos subdivididos en sus correspondientes apartados.

En el capítulo 1 se expone la introducción, en la que se justifica la unidad temática de la presente tesis doctoral y la bibliografía de apoyo. La introducción (subapartado 1.1) enmarca la presente tesis doctoral en los ámbitos donde cobra interés, la industria alimentaria y la salud, define la problemática a resolver, y justifica el interés e importancia de los trabajos realizados. De acuerdo a ello, se exponen los objetivos propuestos (subapartado 1.2), y se describe la estructura de la memoria (subapartado 1.3). El capítulo 2 , consideraciones teóricas, que formará parte de un volumen de una enciclopedia, aborda una revisión bibliográfica exhaustiva sobre los alimentos ahumados. En este capítulo el lector podrá encontrar información extensa sobre la historia de los alimentos ahumados, su rol en la dieta humana, su evolución en los distintos mercados de la economía mundial, los distintos tipos de alimentos ahumados y tecnologías de ahumado, la composición química del humo y los efectos deseados y no deseados que provoca en los alimentos. Entre los efectos deseados se detallan la prolongación de la vida útil y mejora del perfil organoléptico de los distintos alimentos. Entre los efectos no deseados el capítulo describe la contaminación de los alimentos por diversas sustancias tóxicas como las nitrosaminas, las aminas aromáticas heterocíclas y los ! -carbonilos, y se enfoca en la contaminación por HAP.

En el capítulo 3 , se describe de forma general, la metodología experimental, y las diferentes técnicas analíticas utilizadas en la presente tesis doctoral. La metodología utilizada en cada trabajo se detalla específicamente en el artículo científico correspondiente.

Capítulo 1

El capítulo 4 , resultados, es la parte central de la presente memoria de tesis doctoral. En él se presentan los resultados obtenidos, organizados en 5 artículos científicos que abordan el tema principal de la tesis, “el estudio de la contaminación por benzo(a)pireno de chorizos ahumados del Principado de Asturias y las posibles causas para su prevención”. En el capítulo 4.1 se expone la puesta a punto de un método analítico para la determinación de BaP en chorizo ahumado, y su aplicación en chorizos ahumados elaborados en el Principado de Asturias. En el resto de capítulos, se exponen los resultados del estudio de las causas, o variables de proceso, por las cuales el chorizo se contamina por BaP, durante el ahumado. Así, el subapartado 4.2 se enfoca en las variables “tiempo de ahumado y penetración del compuesto en distintas profundidades del producto ”, y los subapartados 4.3 y 4.4 en la influencia y caracterización del uso de distintos tipos de tripa, natural y de colágeno, en el mecanismo de contaminación por BaP (subapartado 4.3), y en la evolución de las propiedades, textura, color y humedad, y contaminación por partículas de humo (subapartado 4.4), del chorizo durante el ahumado. Finalmente, el subapartado 4.5, consta de un artículo, que aborda una revisión bibliográfica exhaustiva sobre el proceso tecnológico de ahumado de productos cárnicos, y el estado del arte en el control de la contaminación por HAP durante el mismo. En esta revisión, el lector podrá encontrar información y conceptos, tales como las referencias históricas, el avance y las modalidades de la tecnología de ahumado de productos cárnicos, el objetivo del ahumado, la formación química de HAP durante el mismo, la evolución de la regulación internacional sobre HAP en productos cárnicos, y los métodos analíticos utilizados por la comunidad científica para su determinación. En la parte central, encontrará una revisión de la presencia de BaP y HAP en productos cárnicos ahumados de diversos países, y su adecuación a la normativa, y por otro lado, estudios científicos sobre las variables del proceso de ahumado que afectan a la contaminación de los productos cárnicos por HAP (de acuerdo al Codex Alimentarius). El artículo concluye con la selección de las variables más importantes a controlar para la prevención de la contaminación por HAP de cárnicos ahumados.

El capítulo 5 expone una discusión general sobre los resultados más importantes encontrados en la presente tesis doctoral, el avance que éstos suponen en el conocimiento científico-técnico y en su aplicación industrial. En el capítulo 6 se exponen las principales conclusiones, que resumen los hallazgos más importantes encontrados en esta tesis doctoral.

Introducción

El capítulo 7 detalla el listado de referencias científicas y bibliografía utilizada en la presente memoria de tesis doctoral, omitiendo, las referencias asociadas a cada capítulo de resultados, las cuales se detallan en el apartado “referencias” de cada publicación correspondiente.

Finalmente, el capítulo 8 , titulado “a nexos ”, expone la difusión de la presente tesis doctoral mediante artículos científicos y comunicaciones a congresos, y el informe sobre el factor de impacto de las revistas científicas de los artículos publicados.

•••••••••••••••••••••••••••••••••••••••••••••••••••••••••••• Consideraciones• •••••••••••••••••••••••••••••••••••••••••••••••••••••••••••• ••••••••••••••••••••••teóricas• • • • • • • • • Capítulo 2

2.•CONSIDERACIONES•TEÓRICAS•

En este capítulo de la memoria se describen los alimentos ahumados. El capítulo introduce la historia del ahumado de alimentos, su rol en la dieta humana y su evolución en los mercados económicos. El capítulo presenta una revisión bibliográfica sobre los las diferentes tecnologías de ahumado y los principales tipos de alimentos ahumados, carnes, pescados y mariscos, quesos, bebidas y especias. Así mismo, se describe la composición del humo y se detallan los efectos deseados y no deseados que presenta en los productos. Entre los efectos deseados se describe la prolongación de la vida útil y la mejora del perfil organoléptico, el color, la textura y el flavor de distintos tipos de alimentos. Por otro lado, se desarrolla en detalle el principal efecto no deseado del ahumado, la contaminación de los alimentos por sustancias tóxicas y cancerígenas. Entre ellas se hace una introducción a las nitrosaminas, las aminas aromáticas heterocíclicas y los •-carbonilos, y se realiza una revisión detallada de los hidrocarburos aromáticos policlíclicos. Se describe la contaminación por PAH de varios tipos de alimentos ahumados, su toxicidad, aspectos legales sobre su regulación, y mecanismos generales para su prevención. El capítulo concluye caracterizando la necesidad y posibilidad de la prevención de los efectos no deseados, manteniendo las buenas propiedades de los alimentos ahumados.

Publicación: Smoked Food. Situación: Pendiente de envío para su revisión.

Consideraciones teóricas

SMOKED FOOD

E. Ledesma a,b , M. Rendueles a & M. Díaz a

a Department of Chemical Engineering and Environmental Technology, University of Oviedo, Faculty of Chemistry, C/ Julián Clavería s/n, 33071 Oviedo, Principality of Asturias, Spain. b Research, Development and Innovation (R&D+i) Department, El Hórreo Healthy Food S.L, C/Las Cabañas 43-45, 33180, Noreña, Principality of Asturias, Spain.

Corresponding author: Tel.: +34 985103439; fax: +34 985 103434.

E-mail address: [email protected] (M. Rendueles), [email protected] (M. Díaz).

ABSTRACT

This chapter is focused on smoked food. It starts by presenting an overview of the history of smoked food, its role on human diet and evolution in economic markets. Then the main types of smoked food and smoking methods are widely described. Next, the composition of smoke and its desirable and non desirable effects on smoked food are characterized in detail. Among the desirable effects there are food preservation and the improvement of the organoleptic profile, colour, texture and flavor of different types of food. On the other hand, the main undesirable effect of smoking is defined in detail. This is the contamination of food by toxic and carcinogenic compounds, standing out polycyclic aromatic hydrocarbons, and others, such as n-nitrosamines, heterocyclic aromatic amines and • -carbolines. Finally, the main conclusions are summarized, showing the necessity and possibility of preventing the non desirable effects and maintaining the good properties of smoked food.

Keywords: Smoked food, smoking methods, food preservation, food flavouring, polycyclic aromatic hydrocarbons, n- nitrosamines, heterocyclic aromatic amines, • -carbolines, food contamination, food control.

Capítulo 2

INDEX

1. History 2. Types of smoked food 2.1 Meat products 2.2 Fish and sea food 2.3 Cheeses 2.4 Beverages 2.5 Spices 3. Food smoking methods 3.1 Direct smoking methods 3.1.1 Traditional cold smoking 3.1.2 Traditional hot smoking 3.2 Indirect smoking methods 3.2.1 Smoked produced by friction generator 3.2.2 Liquid smoke 3.2.3 Electrostatic smoking 3.2.4 Other smoke generation technologies 4. Effect of smoking in food 4.1 Smoke composition 4.2 Food preservation 4.3 Food flavouring 4.3.1 Colour 4.3.2 Texture 4.3.3 Flavour 5. Smoked food toxicology 5.1 Polycyclic aromatic hydrocarbons 5.1.1 Toxicity 5.1.2 PAH contamination mechanism 5.1.3 Regulation 5.1.4 PAH in smoked food 5.1.5 Prevention 5.2 Other toxic compounds 5.2.1 Formaldehyde 5.2.2 N-Nitrosamines 5.2.3 Heterocyclic aromatic amines 5.2.4 •-carbolines 6. Conclusions

Consideraciones teóricas

1. History

Smoked food have had an important historical and economical role in human diet, and are still highly consumed and requested in modern markets and in some developing countries. Food smoking is one of the oldest technological processes which mankind has used since the beginning of time (Tóth, 1982). Probably meat products were the first smoked food produced by humans. It is possible that first humans hung the food over the fire as a protection method against canines, then the preservative action of smoke to prolong food shelf life was probably found (Šimko, 2002, 2009). The first proof of smoking as technological process dates back to 90.000 years ago. The oldest smoking house was discovered by archeologists in a stone age colony located in Zwierzymec, near Krakow in Poland (Möhler, 1978). Smoking process has been used like preservation method along the different ages of men. Food smoking was described in roman cultures in The book of Marcus Cato, 160 B.C, De Agri Cultura, (Leroy, Geyzen, Janssens, De Vuyst, & Scholliers, 2013; Mateo, Caro, Figueira, Ramos, & Zumalacarregui, 2009; Möhler, 1978; Zeuthen, 2007), and in middle age in the book of Marx Rumpolt, 1581 (Möhler, 1978). Romans probably learnt meat smoking methods from the Gauls and Celts. Europeans emigrants export this technique worldwide, including to the Americas, South Africa, and Australia (Leroy et al., 2013), but the smoking process was probably applied in other countries before the colonization. In the age of discovery, smoked food were very useful in long travels by boats. Smokehouses began very popular in every farm and dwelling of USA, and proliferated in the states such as Georgia, Carolinas, Virginia, Tennessee, Missouri and Kentucky. Only the introduction of refrigeration in the 1900´s has stopped smoking as preservation method, especially in meat products (Marianski, Marianski, & Marianski, 2009). Since the 19th century, when rail transport reduced the time from production to market, the production of smoked food decreased (McGee, 2004). Smoked food have had an important role in the diet of people from European countries during World Wars, when food supply was very low. That could explain that smoked meat products are more popular in Europe than in America (Marianski, et al., 2009).

Nowadays smoking is used in developed countries, mainly because of the specific organoleptic profile that it confers to food. Smoking improves food flavour, colour and smell, widely properties demanded by market (Lorenzo, Purriños, García Fontán, & Franco, 2010; Pöhlmann, Hitzel, Schwägele, Speer, & Jira, 2013a; Šimko, 2002; Vaz -Velho, 2003). Smoked food have still a huge impact on the economy of countries. In fact, 30% of the meat products are

Capítulo 2

supposed to be smoked in USA (Marianski et al., 2009), while this figure is 60% in some European countries, like Germany (Frede, 2006). Smoking as preservation method has been replaced by modern methods like controlled drying chambers, liquid smoke (Kostyra & Bary •ko -Pikielna, 2006; Lingbeck et al., 2014; Theobald et al., 2012) and other methodologies for fresh food preservation by means of refrigeration (like chilling, freezing, superchilling), ionising radiation, chemical preservatives and biopreservation, high hydrostatic pressure (HHP) and packaging methods like vacuum packaging, modified atmosphere packaging (MAP), active packaging (AP), antimicrobial packaging, and hurdle technology (HT) (Zhou, Xu, & Liu, 2010).

On the other hand, smoking is still used as preservation method in some developing countries, like Africa and Asia, where a refrigeration chain is not widely established (FAO- Thiaroye, 2015; Vaz-Velho, 2003). For instance, smoking is applied by the artisanal finishing population in Sub-Saharan Africa, which represents the 70% of the whole fishing entrepreneurs in this country. The bulk of all fish caught and all game hunted for food in this country, accounting 80% of the total, are smoke or heat preserved. More than 500 metric tons of smoked fish are exported from West Africa to the United Kingdom each year. This is valued at between 9.3 and 14.9 million United States dollars (Ogbadu, 2014).

2. Types of smoked food

A great number of smoked food have traditionally been produced, and can still be found in the market. Table 1 shows the main groups of smoked food, that are described in this section.

Consideraciones teóricas Table 1. Some types of smoked food. Smoked food Origin/ main use Smoked food varieties Smoked meat products Sausages Austria Vienna sausage Croacia and Serbia Kulen France Andouille, morteau sausage Hungary Hungarian sausage, winter salami Germany Ahle wurst, amsterdam ossenworst, bierwurst, bockwurst, debrecener, knackwurst, knipp, kochwurst, kohlwurst, liverwurst, braunschweiger, mettwurst, pinkel, teewurst Greek Loukaniko Italy Bologna sausage, ciauscolo, lucanica, salami Lithuanian Skilandis Netherlands Rookworst Pennsylvania Lebanon bologna Poland Kielbasa, krakowska, linguiça Portugual Farinheira, chouriço, paínho,paio tradicional, chouriço mouro, cacholeira and morcela. Romania N•dlac sausage Serbia Cajna sausage and sremska sausage, sremska kobasica, Petrovská klobása. Spain Androlla, botillo, chistorra, chorizo, farinato, longaniza, morcilla, morcón, sabadiego, salchichón, sobrasada, smoked dry cured sausage and smoked potato sausage. Sweden Isterband Turkey Sucuk USA Breakfast sausage, half-smoke Ham Bulgaria Elenski but Croatia Pršut England Yule ham France Jambon de Bayonne Germany Ammerländer schinken, black forest ham, eisbein, schwarzwälder schinken, westphalian ham. Italy Smoked prosciutto Portugal Jamón ahumado Romania Jambon Spain Jamón ahumado Sweden Swedish ham smoked USA Country ham, tasso ham

Capítulo 2

Table 1. (continued). Smoked food Origin/ main use Smoked food varieties Smoked meat products Other varieties Africa Smoked pork from Cape Town of smoked meat Brasil Smoked bacon, loin, turkey. products Bosnia and Sebia Suho meso Canada Bacon, oreilles de crises, montreal-style smoked meat China Zhangcha duck Estonia Smoked meat France Brési Germany Bacon, dutch meatloaf, flurgönder, kassler, schäufele Hungary Szalonna Iceland Grjúpán, hangikjöt Indonesia Se'i Irland Bacon Italia Pitina Japon Bacon Kazakhstan Qarta, zhal Korea Jeju black pig Kuwait Smoked meat Malaysia Beef satay Norway Smalahove Pennsylvania Pennsylvania dutch meatloaf Spain Cecina, chosco, smoked tenderloin, smoked jowl. Taiwan Smoked chicken United Kingdom Bacon, gammon, speck, turkey bacon USA Bacon, burnt ends, chaudin, dutch loaf, picnic ham" shoulder, pork jowl Various countries Jerky, meatloaf, pastrami, pickled pigs feet, pork tail, salo

Consideraciones teóricas Table 1. (continued). Smoked food Origin/ main use Smoked food varieties Smoked fish Smoked fish Africa African longfin eel, bokkoms, Ethmalosa fimbriata, smoked Sardinella sp. and anchovies (Anchou guineensis) products Iceland Smoked Salmo Salar Indonesia Cakalang fufu, Ambon’s smoked tuna Japan Katsuobushi (smoked skipjack tuna) Korea Gwamegi Norway Smoked Salmo Salar Philippine Tinapa Scandinavia and Smoked cod roe, smörgåskaviar Finlandia Spain Atlantic salmon (Salmo salar) treated with liquid smoke flavourings Sweden Buckling, Lysekil caviar United Kingdom Arbroath smokie, Finnan haddie, Traditional Grimsby smoked fish (cod and haddock) USA Lox Various countries Atlantic mackerel, Bückling, eel, cod, haddock, halibut, goldeye, herring (Bloater herring, blueback herring, over t he world kipper, craster kipper), mackerel, mussels, oyster, pollock, salmon, sardines, tilapia, scallo, sprats, trout.

Smoked cheese Ireland Ardrahan cheese, Corleggy cheeses, Burren Gold (Gouda style), Gubbeen farmhouse cheese India Bandel cheese Romania Brânz• de co"ule# France Brie Armenia Chechil Russia Chechil England Cheddar cheese, Lincolnshire Poacher cheese, Wensleydale cheese, Orkney (cheddar) Circassia Circassian smoked cheese Spain Gamonéu cheese, Herreno cheese, Palmero cheese, Peña Pelada smoked cheese, Idiazábal cheese, San Simón Da Costa cheese, Liébana cheese, Campoveja cheese, Pría smoked cheese Netherlands Gouda cheese

Capítulo 2 Table 1. (continued). Smoked food Origin/ main use Smoked food varieties Smoked cheese Slovakia Korbá•ik South Africa Kwaito cheese Greece Metsovone, Kashkaval Italy Smoked mozzarella, Provola, Provolone, Ricotta, Scamorza, Caciocavallo Poland Oscypek Slovakia Oštiepok, parenica Serbia Pule Turkey Circassian cheese, Kashar cheese Germany Rauchkäse, reichkäse, caramakase USA Boerkäse Brasil Queijo prato Norwey Smokelet Hungary Szekeley Iran Seretpanir

Smoked beverage Whisky UK (Scotland),Irland Whisky Tea China Lapsang souchong, Assam smoked oolong. Taiwan Lapsang souchong (Tarry souchong) India Mattha (Mate), Earl Grey smoked tea South America Mate Beer Germany Rauchbier Other China Suanmeitang, grappa (Spanish aguardiente de orujo)

Smoked spices America, Hungary, Paprika Turkey and Spain Various Liquid smoke (inventor: Fessmann Gerhard) Other countries Smoked salt, smoked garlic, merquén and chipote

Consideraciones teóricas

2.1 Smoked meat products

Smoked meat products are one of the most important and produced smoked food. They probably were the first smoked food produced by humans. Meat products are an important component of a healthy and well balanced diet (De Castro Cardoso Pereira & Dos Reis Baltazar Vicente, 2013), mainly due to its high protein content (De Castro Cardoso Pereira & Dos Reis Baltazar Vicente, 2013; FAO, 2014a; USDA/HHS, 2010; WHO/FAO, 2003). They are the most valuable livestock products. Thereby, world meat production is projected to double by 2050. In 2015, the average world meat per capita consumption is expected to be 41.3 kg/year. This consumption will be 31.6 kg/year and 95.7 kg/year in developing and industrial countries respectively (FAO, 2013, 2014a). Smoking was an important way to preserve this type of food in the history. Thereby, the most typical smoked meat products are produced in the traditional way.

The table 1 shows some smoked meat products from different countries around the world, that have been studied by researchers (Chiu, Lin, & Chen, 1997; Djinovic, Popovic, & Jira, 2008; Fretheim, 1976; García-Falcón & Simal-Gándara, 2005; Gomes, Santos, Almeida, Elias, & Roseiro, 2013; Hitzel, Pöhlmann, Schwägele, Speer, & Jira, 2013; Jahurul et al., 2013; Ledesma, Rendueles, & Díaz, 2014, 2015a,b; Lorenzo et al., 2010; Lorenzo et al., 2011; Marianski et al., 2009; Martin & Ruiz, 2007; Roseiro, Gomes, Patarata, & Santos, 2012; Santos, Gomes, & Roseiro, 2011; Pöhlmann et al., 2012, 2013a,b; Purcaro, Moret, & Conte, 2009; Reinik et al., 2007; Šimko, 2002; Škaljac et al. 2014; W!grzyn, Grze"kiewicz, Pop#awska, & G#ód, 2006; Wretling, Eriksson, Eskhult, & Larsson, 2010; Yabiku, Martins, & Takahashi, 1993). As the table 1 shows, the most known smoked meat products can be classified in 3 types, sausages, hams and the rest of smoked meat products. Most of the types of smoked meat sausages and hams are produced by Germany, Spain, Portugal and Italia.

2.2 Fish and sea food

Fish production and consumption is growing in the world. Global fish production was 158 million of tones in 2012. The human consumption increase from 9.9 to 19.9 kg per capita, from 1960 to 2012. The 12% of this fish was commercialized smoked, dried, cured or salted, and the rest was used fresh and refrigerated (46%), frozen (29%) and transformed or canned (13%) (FAO, 2014b). The smoking sector is of considerable economic importance to the fish product market,

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accounting for a profit of over US$1,110,000,000 (FAO, 2008). The world production of smoked fish is near 160,000 tons (FAO, 2008).

Table 1 shows some typical kinds of smoked fish from different countries around the world, that have been studied by several researchers (Adekunle & Akinyemi, 2004; Birkeland, Rørå, Skåra, & Bjerkeng, 2004; Brett, Short, McLauchlin, 1998; Cardinal et al., 2001; Gómez- Estaca, Montero, Giménez, & Gómez-Guillén, 2007; Hayward & Mosse, 2012; Ikutegbe & Sikokib, 2014; Ishizaki, Saito, Hanioka, Narimatsu, & Kataoka, 2010; Martinez, Salmerón, Guillén, & Casas, 2007; Montiel, De Alba, Bravo, Gaya, & Medina, 2010; Plahar, Nerquaye-Tetteh, & Annan, 1999; St o•yhwo, Ko•odziejska, & Sikorski, 2006; Yanar, Çelik, & Akamca, 2006).

Salmon and herring are the most important smoked species with a production of around 51,000 and 15,000 tons respectively in 2006. Other important types of smoked fish are cod, tuna, mackerel or trout (Fuentes, Fernández-Segovia, Serra, & Barat, 2010). Fish can be smoked by direct methods, such as traditional smoking, or indirect methods, such as liquid smoke. The main producers of fish smoked in a traditional way are Africa and Asia (FAO, 2014b). As it has been previously said, 80% of the fish captured and processed in Africa is smoked or heat preserved (Ogbadu, 2014). The lost and deterioration of smoked fish are important problems in this country. An special technique of smoking, known as “ technique FAO- Thiaroye” has recently been designed in the city of Senegal, thanks to a project of USA Red Cross and FAO (FAO-Thiaroye, 2015). In Europe, the main producers of smoked fish are the central and oriental Europe countries, especially Poland and the Baltic states (FAO, 2014b). Both direct and indirect smoking methods are applied in these countries.

2.3 Cheeses

Cheese is a dairy product that is available in a wide range of types. Cheese types are produced by using different types of milk, from cow, cattle, sheep, goat, she-ass, etc., and manufacturing processes, including different types of rennets, molds, repining degrees, and smoking processes (Naccari et al., 2009). Cheese has been traditionally smoked in order to prolong its shelf life. The shelf life of milk is short, and smoking became a way to provide good nutrients, as calcium, during all year, when refrigeration chain was not established yet. Nowadays smoking is applied to cheese in order to achieve differentiation (Shakeel-ur-Rehman, Farkye, & Drake, 2003). Cheese can be smoked either by using smoke flavorings or by traditional smoking.

Consideraciones teóricas

However the use of smoke flavorings for the smoking of cheese is not allowed in some countries, such as Spain (Guillén, Palencia, Ibargoitia, Fresno & Sopelana, 2011). Table 1 (Aydinol & Ozcan, 2013; Adhikari, Heymann, & Huff, 2003; Esposito et al., 2015; Garabal, Rodríguez-Alonso, Franco, & Centeno, 2010; Guillén, Ibargoitia, Sopelana, Palencia, & Fresno, 2004; Musullugil & Koca, 2012; Naccari et al., 2009; Palencia, Ibargoitia, Fresno, Sopelana, & Guillén, 2014; Shakeel-ur- Rehman et al., 2003) shows different types of smoked cheeses according to its origin. As table 1 shows, different types of smoked cheeses are produced all over the world. The most common varieties of smoked cheeses are Seretpanir (Iran), Caramakase (Germany), Bandal (India), and Provolone (Italy) (Shakeel-ur-Rehman et al., 2003).

2.4 Beverages

Opposite to other food, the aim of smoking in beverages production has not traditionally been prolong shelf life. The main aim of smoking in the beverages process was flavouring. The main smoked beverages are whisky, tea and beer. Table 1 (Bringhurst & Brosnan, 2014; Buglass & Caven-Quantrill, 2012; Harrison, 2012; Pavsler & Buiatti, 2009; Pincemaille, Schummer, Heinen, & Moris, 2014) shows some types of these smoked beverages.

Whisky is the most famous alcoholic smoked beverage. The most known smoked whisky is produced in Scotch whisky distilleries, especially those located in Islay. The smoking process is applied to whisky during a step often named peating (Bringhurst & Brosnan, 2014). The location from which the peat is extracted is an important factor to give distinctive characteristics to the spirit. By burning of peat, malted barley is dried and some flavouring characteristics are produced, such us peaty, phenolic, smoky, burnt, and medicinal tastes and smells (Bringhurst & Brosnan, 2014). The degree of thermal degradation and decomposition of lignin and other polyphenol components from the plant, have a great influence in the flavour composition of the peat smoke (Harrison, 2012).

Tea (Camellia sinensis) is the most widely consumed beverage in the world, after water (FAO, 2015; Pincemaille et al., 2014). The most known smoked tea is the black tea, in particular Lapsang Souchong. Lapsang Souchong comes from south-eastern China and Taiwan, where is often known as Tarry Souchong. Pine, spruce and bamboo are typical plants used for the smoking of tea. They give a heavy smoky taste and smell to the tea leaves and its infusions (Pincemaille et al., 2014). Other smoked teas are Mate, typical of South American countries such as Paraguay,

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Uruguay, Argentina, Bolivian Chaco and southern Brazil, and Mattha tea, from India. During the manufacturing process for the herbal infusion yerba mate (Ilex paraguariensis), the leaves are dried slowly often using wood smoke (Heck & De Mejia, 2007). The special processing gives mate its typical bitter aromatic and slightly smoky flavor (Schulz, Fritz, & Ruthenschrör, 2014). Mattha may be smoked before serving for flavouring proposes.

Beer is one of the oldest and most popular beverages all over the world (Wunderlich & Back, 2009). There are more than 100 varieties of beer. Depending on the process used, a first classification can be made according to the fermentation process, in top and bottom fermentation beers. Bottom fermented beers are commonly named Lager. Lager is the dominant style in almost all countries, and represents more than 90% of the beer produced worldwide. Top fermentation beers are commonly named Ale, and are very popular in Britain, Germany, Canada ’ s eastern provinces, United States and Belgium (Pavsler & Buiatti, 2009). The most popular type of smoked beer is a top fermentation beer named Rauchbier. This smoked beer is produced in a town of Germany called Bamberg. An historic brewpub named Schlenkerla is especially well known for its smoked Aecht Schlenkerla Rauchbier. Thereby the production of this beer is not very important compared to the whole beer sector. During the production of Rauchbie an aromatic smoke is produced by means of burning beech-wood logs. A smoky bacon flavour and aroma are achieved by exposing the malt to this intense, aromatic smoke. After mixing it with premium-class hops in the brew, it matures in 700 –year –old cellars. The percentage of alcohol of this smoked beer ranges between 4.8% and 6.0% (Buglass & Caven-Quantrill, 2012; Pavsler & Buiatti, 2009).

Finally, other smoked beverages can be found in the world, such as grappa or “aguardiente de orujo”, which smoking process of marc has been studied ( Da Porto, 2012; Da Porto, Moret, & Soldera, 2006), Jamaican rum, the traditional Chinese beverage Suanmeitang, that is made of sour plums (specifically, smoked Chinese plums), rock sugar, and other ingredients such as sweet osmanthus.

2.5 Spices and flavourings

Some types of smoked spices are shown in table 1 (Doymaz & Pala, 2002; Gallardo- Guerrero, Pérez-Gálvez, Aranda, Mínguez-Mosquera, & Hornero-Méndeza, 2010; Lingbeck et al., 2014; Mateo, Aguirrezábal, Domínguez, & Zumalacárregui, 1997; Ramesh, Wolf, Tevini, & Jung,

Consideraciones teóricas

2001; Turhan, Turhan, & Sahbaz, 1997). The most known and studied smoked spices and flavourings are paprika and liquid smoke. Other less known types of smoked spices are smoked salt, smoked garlic and smoked chili peppers, like merquén and chipote.

2.5.1 Paprika

Paprika is a red powder made from grinding the dried pepper pods of some varieties of Capsicum annuum L (Mateo et al., 1994; Reineccius, 1994). This natural food product is commonly used as spice and natural colorant to provide redness to other food, such us meat products and commercial sauces (Attokaran, 2011). Although paprika is original from America, it is also produced in Europe, particularly in Hungary, Turkey and Spain (Palacios-Morillo, Jurado, Alcázar, & de Pablos, 2014). Some kinds of paprika, such as the Spanish “pimentón de la Vera ”, are dried by traditional smoking process. During smoking, logs are burnt and the ripe red fruits of the pepper are slowly dehydrated by means of the heat and smoke. Smoking time ranges between 7 to 10 days (Gallardo-Guerrero, et al., 2010) or 12 to 15 days (Mateo et al., 1997). Temperature in the chamber is about 40ºC during the first 5 days, and 60ºC from 5 days to the end of the process. The final water content of the product is below 15% (Mateo et al., 1997). Drying process has an important role on the composition of final paprika, in its carotenoids content and non-enzymatic browning (Lee & Kim, 1989), colour, l-ascorbic acid and sugar retention (Sigge, Hansmann, & Joubet, 1999) or colour (Carbonell et al., 1986; Doymaz & Pala, 2002). The dehydration and drying kinetics of red pepper under different pretreatments and drying conditions have been previously studied (Doymaz & Pala, 2002; Ramesh et al., 2001; Turhan et al., 1997). Therefore, smoking process should be optimized in all cases, in order to obtain good quality characteristics of the final paprika.

2.5.2 Liquid smoke

Liquid smoke is a more actual type of food flavouring. Liquid smoke is mainly applied to meat products, fish and poultry, but it is also applied to non-meat food such as cheese, tofu and even pet food (Lingbeck et al., 2014). Liquid smoke is highly used in marinades, sauces or brines (Lingbeck et al., 2014; Rozum, 2009). In addition, liquid smoke exhibits antimicrobial activity against Listeria (Gedela, Gamble, Macwana, Escoubas, & Mariana, 2007; Martin et al., 2010; Messina, Ahmad, Marchello, Gerba, & Paquette, 1988), Escherichia coli (Van Loo, Babu, Crandall, & Ricke, 2012), Staphylococcus aureus and staphylococcal enterotoxins (Lingbeck et al., 2014;

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Taormina & Bartholomew, 2005). During its production, liquid smoke is filtered and subjected to fractionation and purification processes to remove toxic and carcinogenic particles and compounds. Therefore, its use is generally considered to be of less health concern than the traditional smoking process. However, the possibility of broader applications of smoke flavorings compared to conventional smoking has to be taken into account in safety assessments (EC No 2065/2003; Lingbeck et al., 2014). Liquid smoke is more environmentally friendly, faster, more specific and easier of application than traditional smoking, and allows good reproducibility of desired characteristics in the end product (Lingbeck et al., 2014; Suñen, Fernandez-Galian, & Aristimuño, 2001).

3. Food smoking methods

The traditional and uncontrolled method of food smoking by means of biomass fires has been improved, and introduced in some modern developed countries. However, the modern techniques are still very extensive to be used in some sectors and developing countries.

Food smoking methods and techniques have been classified and described in several works (Ahmad, 2003; Möhler, 1978; Šimko, 2009; Vaz-Velho, 2003; Woods, 2003), based on the temperature of the smoke, the location of smoke generation with respect to the position of the foodstuff, and the device used for generating smoke. In this chapter, the different smoking methods are classified in two main groups: direct and indirect smoking.

3.1 Direct smoking methods

During direct smoking, smoke is produced in the same chamber where the meat is processed (CAC/RCP 68/2009). Direct smoking methods mainly comprise traditional techniques. The traditional smoking method consists of direct thermal degradation of wood to produce smoke (Ahmad, 2003). This definition can be extended, as others kinds of fuels were erroneously used in the past all over the world. In fact, humans used to burn any kind of waste in bonfires to produce smoke, such as food waste (coconut shells, ears of corn, fruit stones, etc.) and even newspapers or furniture (wood treated with paint) (CAC/RCP 68/2009). Various unhealthy compounds are formed if the correct fuel is not used, especially PAH. Direct smoking methods can be classified according to the temperature of the smoke.

Consideraciones teóricas

3.1.1 Traditional cold smoking

During cold smoking, wood is burned and smoke is produced. Meat products are hung from shelves placed above the hearth, located in a grilled floor through which the smoke passes. Smoking chambers are usually large. When burning finishes, the fire is not poked and the smoke cools. According to different authors, the required temperature in a smoking chamber to achieve cold smoking conditions should be below 20°C (Möhler, 1978), between 15 - 25°C (Šimko, 2009; Woods, 2003), or below 30°C (Ahmad, 2003). Raw hams (Möhler, 1978; Šimko, 2009), heat fermented untreated products like salami (Šimko, 2009) and other stuffed meat products, like chorizo, are usually produced by cold smoking.

3.1.2 Traditional hot smoking

During traditional hot smoking, the chamber is heated by the burning of wood in a similar process to a typical old baking oven. Once placed inside the chamber, the meat is heated and dried by embers of burnt wood. Sawdust is then introduced into the chamber and the fire is stoked with the aim of producing a large amount of smoke (Möhler, 1978). Temperatures of 130°C in the smoke and 80°C in the meat are needed in hot smoking (Ahmad, 2003; Möhler, 1978), although some authors specify lower temperatures, between 55 and 80°C (Woods, 2003).

3.2 Indirect smoking methods

Indirect smoking comprises a number of new methods that help reduce PAH contamination of meat products. These are summarized below.

3.2.1 Smoke produced by a friction generator

Primitive tribes produced smoke during the discovery of fire by means of the uninterrupted friction of wood on wood. This may well have provided the inspiration for friction smoke generation methods. The first development of this technique for producing smoke was first developed as a modern technology by Rasmussen and Rasmussen (1961) and was protected by US patent 3.001, 879. Since then, new models have been introduced. Nowadays, smoking chambers are designed to control all the processing steps, including preheating and reddening, friction smoke generation, smoke evacuation and drying. These steps are repeated in cycles, the number and duration of which depend on the type of meat product. Typical smoke generation

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values are 20-second intervals of continuous friction of a gearwheel with wood followed by a pause of between 70 and 175 seconds. During this process, a temperature range between 180 and 380°C is reported by Varlet et al. (2007). The process, adapted for the production of a typical Spanish meat product called chorizo, includes 5 cycles of 30 minutes of preheating (150 minutes), reducing the temperature from 18 to 2°C and the humidity from 95 to 90%, 6 hours of drying at 25°C and 90% humidity, 41 cycles, consisting of 10 minutes of smoking, 3 minutes of smoke evacuation, and 20 minutes of drying at 25°C and 90% humidity, and a final step of 3 cycles of 8 hours of drying (24 hours) at 80% humidity, decreasing the temperature from 23 to 18°C. According to Pöhlmann et al. (2013a), Frankfurter-type sausages are exposed to reddening for 10 min at 52°C, drying for 12 min at 56°C and friction smoking for 26 to 40 hours, followed by a final step of scalding at 75°C for 25 min. With friction smoke generation, operation time and wood requirements are reduced and production is controlled and optimized. Furthermore, meat industry safety is increased, as PAH production is very low (Pöhlmann et al., 2013a), workers’ health is improved by preventing fire hazards, product weight losses are reduced, product flavor is enhanced by avoiding the concealing of the taste of the ingredients, product shelf life and quality are suitable and product standardization and homogenization is made possible.

3.2.2 Liquid smoke production

The development of smoke flavourings dates back to the late 19th century (Fessmann, 1972; Miler & Kozlowski, 1969). It was developed with the aim of replace the traditional smoking process (Theobald et al., 2012). The flow diagram of typical liquid smoke production has been recently described by Lingbeck et al. (2014). Nowadays, liquid smoke is produced by condensing wood smoke formed by the controlled, minimal oxygen pyrolysis of sawdust or wood chips. The wood is placed in large retorts where intense heat is applied, causing the wood to smolder (not burn), releasing the gases seen in ordinary smoke. These gases are quickly chilled in condensers, thus liquefying the smoke. The liquid smoke is then forced through refining vats and subsequently filtered to remove toxic and carcinogenic impurities containing PAH. Finally, the liquid is aged for mellowness (Lingbeck et al., 2014). An outline scheme for the preparation of a PAH-free liquid smoke was described by Ahmad (2003). The use of liquid smoke is considered to be healthier than the use of smoke obtained in the traditional way. However, the possibility of broader applications of smoke flavorings compared to conventional smoking has to be taken into account in safety assessments (EC No 2065/2003; Lingbeck et al., 2014).

Consideraciones teóricas

3.2.3 Electrostatic smoking

In this type of smoking, the product is positioned in a continuous tunnel between live electrical wires that are charged to between 20 and 60 kV. Smoke passing through this system is charged according to its phase (smoke is a two-phase system, particulate and vapor), and smoke components can precipitate on the oppositely charged food surface (Vaz-Velho, 2003; Woods, 2003). The movement of gas and liquid in smoking chambers has barely been studied (Pinilla, Díaz, & Coca, 1984). In order to ensure sedimentation of the smoke components on the surface of the product, the smoking step is usually followed by infrared irradiation (Möhler, 1978). This process helps to avoid PAH contamination of foodstuffs.

3.2.4 Other smoke generation technologies

Other well-known smoke generation technologies include steam, fluidization, touch and smoldering smoke generators. Steam smoke is produced by passing superheated steam through chopped wood, inducing pyrolysis. The resulting smoke passes through the smoking chamber, being cooled to 80°C (Prändl, 1994; Tóth & Potthast, 1984). According to Müller (1982), the steam smoke generation temperature varies between 450 and 650°C. A fluidization smoke generator allows pyrolysis of wood shavings suspended in air that has been previously heated to 300-400°C. Pyrolysis is carried out within a reaction chamber and smoke and solid particles are separated on passing through a cyclone refiner (Klettner, 1979; Nicol, 1960; Prändl, 1994; Tóth & Potthast, 1984). The schematic representations of the different smoking generators are reported by Prändl (1994). Smoldering, steam and touch smoke generation systems for the production of Frankfurter-type sausages are described in detail by Pöhlmann et al. (2012, 2013a). In these technologies, smoking conditions are highly controlled and optimized, including different smoke densities (light, medium, and intensive), temperatures of smoke generation (ranging between 300 and 520°C), ventilator speeds (750, 1500, and 3000 rpm) and exposure times to smoke (from 3 to 40 minutes). The control of smoking conditions in these smoke generation technologies allows the prevention of final contamination of meat products with PAHs (Pöhlmann et al., 2013a).

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4. Effect of smoking in food

4.1 Composition of smoke

The effect of smoking in food is defined by its composition. Smoke used in food production consists of a suspension of solid particles in a gaseous phase of air, carbon oxide, carbon dioxide, water vapor, methane, and other gases, making up an aerosol (Ahmad, 2003; CAC/RCP 68/2009 ; Šimko, 2009 ; Woods, 2003). The size of these particles generally ranges between 0. 2 !m and 0. 4 !m, and the total range is 0.05 !m -1 !m ( CAC/RCP 68/2009). The main factors affecting smoke composition are the type of wood, the wood combustion temperature, the amount of water vapor available (or smoke house humidity), the amount of oxygen present, the effect of air flow rate, and the time of smoking (Woods, 2003). Temperature is the most important factor, and the constituents of wood, hemicellulose, cellulose and lignin, react at different temperatures (Ahmad, 2003; Woods, 2003). Table 2 summarizes the temperatures of decomposition of wood constituents (Ahmad, 2003; Woods, 2003).

Table 2. Temperatures of decomposition of wood constituents

Temperature (°C) Thermochemical conversion processes of wood constituents Up to 170 Drying 200-260 Pyrolysis of hemicelluloses 260 –310 Pyrolysis of cellulose 310 –500 Pyrolysis of lignin

Up to 1100 chemical compounds have been identified (Wilms, 2000). Wood smoke is composed by over 400 volatile components comprising 48 acids, 22 alcohols, 131 carbonyls, 22 esters, 46 furans, 16 lactones, 75 phenols, and some 50 miscellaneous compounds (Woods, 2003). Smoke compositions obtained from different types of wood have been described in detail (Ahmad, 2003; Hitzel et al., 2013; Stumpe- V"ksna et al., 2008).

Consideraciones teóricas

Some main components of smoke condensates are shown in table 3 (Ahmad, 2003). Some different groups of smoke components are classified in table 4 (Ahmad, 2003; Guillén & Manzanos, 2002; Kostyra & Bary•ko -Pikielna, 2006; Möhler, 1978; Ojeda, Barcenas, Pérez- Elortondo, Albisu, & Guillén, 2002; Woods, 2003).

Table 3. Composition of a smoke condensate (Ahmad, 2003). Components Percentage (%) Phenols 0.07 Formaldehyde 0.12 Formic acid 0.38 Aldehydes of higher molecular weight 0.57 Ketones 0.67 Methanol 0.96 Acetic acidand acids of higher molecular 1.71 Weight Extracts from activated charcoal 4.08 Residue 4.21 Tar 4.81 Water 82.42 Total 100%

As table 4 shows, smoke contains around 20 groups of chemical compounds. However, these can be divided in 2 main groups depending on their desirable or non-desirable effects in the end product. The desirable compounds are responsible of good food preservation and flavouring, while the non desirable compounds are responsible of concerning about smoked food toxicity. These effects of smoking are described in the next sections.

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Table 4. Chemical composition of smoke and technological effect. MAIN GROUPS OF COMPOUNDS IN SMOKE EFFECT ON MEAT PRODUCTS

Group 1 Saturated and unsaturated aliphatic Low effect due to their low CH series compounds hydrocarbons (paraffin and olefins). reactivity Group 2 Aromatic hydrocarbons (benzol, Negative effects: CH series compounds polyphenol). Bad taste Group 3 Methyl alcohol: precursor of formaldehyde Non-desirable, toxic

COH 2 series compounds: and formic acid. Aliphatic compound s with Ethyl alcohol, propyl alcohol and iso propyl Desirable one hydroxyl, alcohol and alcohol: oxidation to produce carbonyls and ethers: acids. Butyl alcohol and amyl alcohol. Desirable (oil aroma) Fenyl alcohols (benzyl alcohols, phenethyl Desirable (smell of roses) alcohol) Group 4 Guaiacol, methylguaiacol, cresol and Desirable, preservatives and

COH 2 series compounds: xinelone. flavoring Aromatic compounds with Phenol Non desirable, bad taste one hydroxyl, phenol Phenol derivates: Desirable in low quantities Orthocresol, meta cresol, para -cresol Good effects in preservation, Xylenols smoke smell and color Thymol Strong antimicrobial attributes, biocide Fungicide effect Preservative Thyme aroma Group 5 Formaldehyde Desirable COH compoun ds Harden ing of natural casing (carbonyls): Aliphatic Connective tissue aldehydes Bactericide Food preservative Group 6 Benzaldehyde Desirable, COH compounds Bitter almond arom a (carbonyls): Aromatic aldehydes Group 7 Acetone and unsaturated long-chain Undesirable aroma CO compounds : Aliphatic compoun ds. ketones Group 8 Acetophenone Hay smell CO compounds: Hydrindenes Soft aroma Aromatic ketones Group 9 Formic acid and acetic acid. Desirable for: Color setting, COO Compounds: Alyphatic Good smell.Bactericidal effect carboxylic acids Butyric acid, valerianic acid, caproic acid, Smell enanthic acid, caprylic acid, pelargonic acid. Unsaturated carboxylic acid (crotonic and Desirable for color tiglic) No desirable for taste

Consideraciones teóricas

Table 4. (continued). MAIN GROUPS OF COMPOUNDS IN SMOKE EFFECT ON MEAT PRODUCTS Group 10 Benzoic acid Specific action against COO compounds: Aromatic Salycilic acid, gallic acid, toluic acid, phthalic the aerobic sporadic carboxylic acids acid, isophthalic acid, terephthalic acid germ of the genus bacillus which contaminates meat products Group 11: Dihydroxybenzole Aromatic polyvalent Guayacol hydroxy compounds Group 12: Acetol Desirable Hydroxy -oxo- compounds: Reaction with meat Ali phatic h ydroxyaldehydes and proteins to develop the hydroxy ketones. characteristic color of smoke Group 13: Salicylaldehyde (2-hydroxybenzaldehyde), Desirable: intense aroma Hydroxy -oxo- compounds: 4-Anisaldehyde, Vanillin and and flavor of vanilla and Aromatic compounds, phenol coniferaldehyde conifers aldehydes and phenol ketones Group 14: Glyoxal Casing hardening Compounds with various oxo - Diacetyl Margarine and bread groups: color and smell Di aldehydes and Di ketones Group 15 Maleic acid Desirable for color: Compounds with 2 or more pigment carboxyl groups: Saturated and unsaturated dicarboxylic acids Group 16 Pyruvic acid, levulinic acid Oxo - carboxy compounds : Keto acids Group 17 Pyrrole, pyrazine, indole, carbazole Darkening of color Nitrogenous organic compounds

Group 18 Cyclotene Food aroma Non -aromatic cyclic compounds of C Group 19 Furan Polymerization to obtain Heterocyclic compounds dark pigments. Good Lactone ( Maltol ) aroma. Flavor enhancer Group 20 Benzo(a)pyreno, PAH 4 ( •PAH: Non-desirable in food Polycyclic aromatic Benzo(a)pyren e, benz(a)anthracene, science: hydrocarbons (PAH) benzo(b)fluoranthene and chrysene. Toxic, carcinogenic

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4.2 Food preservation

Preservation has been the main objective of food smoking during several years, when the refrigeration chain was not established yet (Marianski et al., 2009), and it is still the main aim in some developing countries (FAO-Thiaroye, 2015; Vaz-Velho, 2003). As table 3 shows, the main component of smoke is water, so the reason of the preservation quality of smoking is not only this high component of smoke (Möhler, 1978). The main ways of smoking for food preservation are drying and the presence of some desirable components in smoke. During drying the water activity of the food decrease. Besides some components, such as thymol, formaldehyde, formic, acetic and benzoic acids, orthocresol, meta-cresol, para-cresol, guaiacol, methylguaiacol, cresol and xinelone have a desirable bactericidal, antimicrobial, biocidal, fungicidal and preservative effects. Both properties limit the growth of some types of microorganisms (Vaz-Velho, 2003). The antimicrobial activity of the different components of liquid smoke has been reviewed (Lingbeck et al., 2014). Some phenolic compounds, especially isoeugenol, in addition with the acids, have an antimicrobial activity on L. monocytogenes (Suñen, 1998; Young & Foegeding, 1993). Not all the phenols have the same property, because phenol concentration is not indicative of the antimicrobial activity against L. monocytogenes (Suñen et al., 2001). On the other hand, organic acids and carbonyls have been found to have more antilisterial and bacteriostatic properties than phenols (Milly, Toledo, & Chen, 2008; Montazeri, Himelbloom, Oliveira, Leigh, & Crapo, 2013). Liquid smoke has demonstrated to be bactericidal against Salmonella Typhimurium (Kim, Kang, Park, Nam, & Friedman, 2012) and E. coli (Van Loo et al., 2012). Liquid smoke has also been found to reduce Staphylococcus aureus and staphylococcal enterotoxins in food (Taormina & Bartholomew, 2005).

4.3 Food flavouring

Nowadays food flavouring is the main aim of smoking in the developed countries. A controlled process of smoking has a desirable effect in the food colour, texture, smell and taste.

4.3.1 Colour

Smoking is applied to improve the colour of the food. Several studies are focused on the changes on the colour parameters, lightness (L*), redness (a*), yellowness (b*), browning index (BI), hue angle, chroma, and total colour change (•E), of different food during smoking, even with

Consideraciones teóricas

different smoke generation methods (Pöhlmann et al., 2013a). These studies include, smoked meat products, such as chorizo (Gimeno, Ansorena, Astiasarán, & Bello, 2000), a turkish dry- fermented sausage named Sucuk (Bozkurt & Bayram, 2006), Frankfurter-type sausages (Pöhlmann et al., 2013) or smoked blood sausages (Silva et al., 2013), smoked fish products, such as smoked Atlantic salmon (Salmo salar L.) (Birkeland, Bencze Rørå, Skåra, & Bjerkeng, 2004; Cardinal et al., 2001) and smoked cheeses, such as smoked Cheddar and Swiss cheeses (Hendrick, Bratzler, & Trout, 1960; Riha & Wendorff, 1993). The common characteristic of any smoked food is a decrease in L* (lightness). The main changes in the colour of any type of food are produced in their external surfaces. It has been found that the greatest number of soot particles is deposited in the external surface of the smoked food (Ledesma et al., 2015b). This fact explains the lightness decrease during food smoking.

4.3.2 Texture

Smoking has an important effect on food texture. Softness or hardness can be considered a good or a bad attribute, depending on the type smoked food. For instance, softness or tenderness can be a positive attribute in some types of chorizo (like “chorizo asturiano”) , while in others (like ‘‘chorizo de Pamplona”) it can be considered a defect (Gimeno, Astiasarán, & Bello, 1999; Spaziani, Del Torre, & Stecchini, 2009). The final texture of the food can be defined by controlling the smoking time and other parameters of the manufacturing process. Some studies have been conducted with this purpose, and in different types of smoked food, mainly in meat products (Kim et al., 2014), fishes (Birkeland et al., 2004; Cardinal et al., 2001), and cheeses (Adhikari et al., 2003). The main determinants of smoked food texture are the extent and rate of water loss, the fat content of the product and its distribution, the extent of denaturation of structural and connective tissue protein, and the extent of autolysis, particularly proteolysis (Woods, 2003). Temperature of smoking also has an important role in the texture of the final product. The hardness of the food generally increases with the smoking time and the temperature. European cold-smoked products generally have a soft and tender texture, while the equivalent hot-smoked products have a hard dry surface with softer interior (Woods, 2003).

4.3.3 Flavour

Flavour is a sensory impression perceived by means of at least three different senses: aroma perception in the olfactory epithelium, taste perception predominantly on the tongue and

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chemesthesis of ‘irritants’ in the areas of the nose, eyes and mouth (Methven, 2015). Smoke components and smoking temperature, time and technique have an important effect on food flavour.

Among all the groups of compounds produced during wood combustion, some flavouring compounds (Möhler, 1978) are cited in table 4. These include butyl alcohol and amyl alcohol (oil aroma), fenyl alcohols, like nenzyl alcohols and phenethyl alcohol (smell of roses) and benzaldehyde (bitter almond aroma) (Möhler, 1978). Several studies about the volatile compounds in smoke flavourings, its sensory characterization and descriptors have been done (Kostyra & Bary!ko -Pikielna, 2006; Ojeda et al., 2002; Pino, 2014). The aroma associated with a specific compound produced during smoking is cited in some works, such as Möhler, 1978. However, while the single flavouring volatile compounds can be separated, identified and quantified by means of instrumental analysis, its sensory analysis and characterization is more problematic. The aroma, taste or smell of a smoke flavouring cannot be related only with one compound (Kostyra & Bary !ko -Pikielna, 2006). However, some compounds seem to have greatest effect, and have been selected as sensory descriptors and standard references of the smoke flavourings (Ojeda et al., 2002), such as nerolidol for fruity, ethylbenzene for combustible, propionic acid for sharp, geraniol for floral, cyclotene for caramellic, maltol for sweet, isobutyric acid for pungent, acetic acid for acidic, thymol for wood, guaiacol for medicinal, eugenol for spice/aromatic herb, and 1-Octen-3-ol for musty flavors (Ojeda et al., 2002). Carbonyls contribute significantly to smoke-curing aroma, but phenols have been confirmed as the main (but not exclusive) contributor to that specific flavor. Phenol, p-cresol and ocresol (phenolic compounds) and cycloten and 3-methylocyclopenten (carbonyls) seem to play the most important role in smoke-curing aroma. Syringol and its derivatives do not contribute to the odour identified as typical for freshly smoked product (Kostyra & Bary!ko -Pikielna, 2006). Besides, the amount of the compound must be enough to reach the flavor threshold of the consumer (Woods, 2003).

The flavor and smell of smoked food is affected by the smoking temperature, time and technique. The smoky flavor and smell are common characteristics of all smoked food. However, specific organoleptic profiles of different types of smoked food have been described. Among the smoked meat products, interesting works have been done in the sensory evaluation of Frankfurter-type sausages. The odour, flavor and complete sensory evaluation of Frankfurter- type sausages smoked by means of smouldering, steam, friction, and touch smoking methods

Consideraciones teóricas

increases with smoking density (from lightly to intensively and medium) and time (from less than 30 minutes to more than 30 minutes) (Pöhlmann et al., 2012, 2013a). The ventilator velocity or the moisture of the beech wood chips have not significant influence on the sausages flavor (Pöhlmann et al., 2012). Temperatures bellow 500ºC and higher than 750ºC produce excessive and insufficient smoke flavours in sausages, respectively (Pöhlmann et al., 2012). Interesting studies about the organoleptic profile of smoked fish have been done. The intensity of smoke odour and flavor of smoked salmon is affected by the smoking temperature or technique. A higher temperature increases the deposit of smoke compounds, and the smoky flavor. Special toasted bread odour and low flavour intensity is produced when this fish is smoked by the electrostatic technique (Cardinal et al., 2001). A model to define the parameters affecting cold- smoked salmon odour and flavour intensity has been proposed (Cardinal et al., 2004). Among all the selected parameters, including remaining time, phenol, lipid, total volatile basic nitrogen, trimethylamine, and salt contents, phenol content has the major influence on smoke odour and flavor of cold-smoked salmon (Cardinal et al., 2004). Several studies about smoked cheeses and beverages flavour have been done. Smoked Cheddar cheeses are characterized by specific smokey and skunky flavors, compared to non smoked Cheddar cheeses. Non smoked Cheddar cheeses exhibited higher intensities of “cooked,” “whey,” “milkfat,” “sweet” and “nutty” flavors than the smoked cheeses (Shakeel-Ur-Rehman et al., 2003). Distinctive smoky and medicinal aromas are conferred to the whisky when the malt is smoked (Bringhurst & Brosnan, 2014; Jack, 2012).

5. Smoked food toxicology

Smoking improves food color, texture, flavor and final value, prices can be reduced and shelf life can be extended (FAO, 2014a; Ledesma et al., 2015b). However, non-desirable substances can be produced during smoking, damaging the final composition of food. The main toxic compounds on smoked food are discussed in this section.

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5.1 Polycyclic aromatic hydrocarbons

5.1.1 Toxicity

Polycyclic aromatic hydrocarbons (PAH) are the best known toxic substances on smoked food. Research about PAH is focused on its carcinogenic activity. The first evidence of PAH carcinogenic activity was discovered in 1775 by Percival Pott of St Bartholomew’s Hospital in London. He noticed that sweeps who cleaned the chimneys removing soot developed cancer (Šimko, 2002) . Since that moment several studies have been done, and the carcinogenic, mutagenic and bioaccumulative capacities of PAH have been reported by the Food and Agriculture Organization of the United Nations (FAO), the World Health Organization (WHO), (WHO, 2006), the International Agency for Research on Cancer (IARC), the European Scientific Committee on Food (SCF), (SCF, 2002), the European Food Safety Authority (EFSA) and the US Environmental Protection Agency (EPA) and the Department of Health and Human Services (ATSDR, 1995; ATSDR, 2009).

5.1.2 PAH contamination mechanism

The mechanism of food contamination by PAH during smoking process has been described in detail (Ledesma et al., 2015b). Smoked food can be PAH contaminated by three ways: “Pre -contamination” of raw materials due to atmospheric pollution ( CAC/RCP 68/2009), food technology processes other than smoking, such as packaging, drying, grilling, roasting (Chung et al., 2011), baking, barbecuing (Kazerouni, Sinha, Hsu, Greenberg, & Rothman, 2001) and frying, and mainly during the smoking process. During smoking the biomass conversion takes place. About 750°C tar aerosols containing PAH are produced (Basu, 2010; Li et al., 2009; Liu et al., 2013; McGrath, et al., 2001; Simoneit, 2002). Then PAH are transported through the smoking chamber and reach products contaminating food (CAC/RCP 68/2009; Ledesma et al., 2015b). Moreover, the fat content of the food falls down onto the fire, thereby increasing the PAH content of the smoke (Chung et al., 2011; Janoszka, Warzecha, Blaszczyk, & Bodzek, 2004). On the other hand, some fat already contaminated falls onto other food, increasing its final PAH content (CAC/RCP 68/2009; Viegas, Novo, Pinto, Pinho, & Ferreira, 2012). The greatest amount of soot particles and PAH is deposited in the external surfaces of food, such as the rind of cheese (Guillén & Sopelana, 2004) or the casing of meat products (Ledesma et al., 2014, 2015b; Santos et al., 2011), then the PAH migrate into the food. Natural casing morphology and physical

Consideraciones teóricas

characteristics (Ledesma et al., 2015b) facilitate PAH contamination and penetration into the smoked meat products.

5.1.3 Regulation

To protect consumers against PAH intake from diet, the European Commission (EC) has adopted several regulations over the last 10 years. The names, abbreviations, relative molecular weights and chemical structures of the 16 priority polycyclic aromatic hydrocarbons (PAH) regulated food products by the European Union are cited in the table 5.

In accordance with the European Scientific Committee on Food (SCF) (SCF, 2002), PAH levels in food have been regulated via EC Regulation No 208/2005 and European Union (EU) Commission Regulations No 1881/2006 and No 835/2011. In accordance with EU regulation No 835/2011 (table 6) two dates must be considered as regards PAH contamination in smoked food since 1/9/2014, a separate level for benzo(a)pyrene (BaP), the main traditional marker, and a new maximum level for the sum of four substances known as “PAH4 ”, BaP, benzo(a)anthracene, benzo(b)fluoranthene and chrysene (EC, No 835/2011). The regulation should continue be reviewed. A recent study by Lorenzo et al. (2011) reports that BaP is still a good marker for the sum of 15 PAH as well as for 7 PAH classified as probable human carcinogenics by the USEPA in ‘chorizo gallego’. There is not a European PAH limit for some smoked food, such as cheese, beer, whisky and some spices, such as papr ika. The Spanish law established a BaP limit of 10 !g/kg in the smoked cheese rind (BOE, 1985; Guillén, Palencia, Sopelana, & Ibargoitia, 2007), and the US EPA established a BaP limit of 0.2 !g/kg in ambient water (ATSDR, 2009; EPA, 2013). On the other hand, the regulation establishes PAH limits for non smoked food, such as oils (including coconut oil) and fats, cocoa beans and derived products, processed cereal-based food and baby food for infants and young children, and infant formulae and follow-on formulae, including infant milk and follow-on milk.

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Table 5. Name, abbreviations, relative molecular weights and chemical structures of the 16 polycyclic aromatic hydrocarbons (PAH) regulated in meat products by the European Union.

PAH compound Abbreviation Molecular weight Chemical structure

Benzo(a)pyrene BaP 252

Benz(a)anthracene BaA 228

Benzo(b)fluoranthene BbF 252

Benzo(j)fluoranthene BjF 252

Benzo(k)fluoranthene BkF 252

Benzo(g,h,i)perylene BghiP 276

Chrysene Ch 228

Cyclopenta(c,d)pyrene CPP 226

Dibenz(a,h)anthracene DBahA 278

Dibenzo(a,e)pyrene DBaeP 302

Dibenzo(a,h)pyrene DBahP 302

Dibenzo(a,i)pyrene DBaiP 302

Dibenzo(a,l)pyrene DBalP 302

Indeno(1,2,3cd)pyrene IP 276

5-methylchrysene 5MeCh 242

Benzo(c)fluorine BcF 216

Consideraciones teóricas

Table 6 . Maximun legal limits for benzo(a)pyrene and PAH4 in smoked foods fixed by EC Regulation 835/2011

Maximum levels (•g/kg) Smoked food Benzo(a)pyrene PAH4 a 5.0 b 30.0 c Smoked meat and smoked meat products 2.0 d 12.0 d Muscle meat of smoked fish and smoked fishery products (The maximum level for smoked crustaceans applies to muscle meat from appendages and abd omen. In case of 5.0 b 30.0 c smoked crabs and crab -like crustaceans (Brachyura and Anomura) it applies to muscle meat from appendage) 2.0 d 12.0 d

Smoked sprats and canned smoked sprats (Sprattus Sprattus). Bivalve molluscs (fresh, chilled or frozen) 5.0 30.0 Heat treated meat and heat treated meat products sold to the final consumer

Smoked bivalve mollusks 6.0 35.0

a PAH4: Sum of benzo(a)pyrene, benz(a)anthracene, benzo(b)fluoranthene and chrysene b until 31/8/2014 c from 1.9.2012 until 31.8.2014 d from 1/9/2014 on

5.1.4 PAH in smoked food

Humans are exposed to PAH due to environmental contamination, but the main source of PAH is diet (Falcó et al., 2003; Ibáñez et al., 2005; Lodovici, Dolara, Casalini, Ciappellano, & Testolin, 1995; Phillips, 1999), contributing to more than 70% of total exposure in nonsmokers (Gilbert, 1994; McGrath, Wooten, Geoffrey Chan, & Hajaligol, 2007). The PAH contamination of smoked food has been studied for a long time. Table 7 shows recent data about PAH in different types of smoked food around the world, including smoked meat products, fish, cheese and some teas.

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Table 7. PAH in smoked food PAH ( •g/kg) World Smoked Food BaP PAH Reference Maximun Minimiun Maximun Minimun Smoked meat products from Africa 10.02 <0.1 Olatunji et al., 2014 smoked pork smoked beef and chicken Estonia 31.20 <0.3 PAH a: 8.0 PAH a: 5.7 Reinik et al., 2007 smoked meat products various products smoked ham smoked chicken Italy 0.8 <0.05 Purcaro et al., 2009 smoked Pitina smoked bacon, smoked pork and beef meats Latvia 6.03 <0.05 PAH4: 34.65 PAH4: 0.15 Rozent le!et!al.,!2015 smoked pork various products smoked pork speck smoked pork Portugal 0.63 0.32 PAH4: 6.94 PAH4: 1.84 Santos et al., 2011 smoked Paínho smoked morcela smoked chouriço smoked morcela mouro Republic of Korea 0.08 0.01 PAH b: 1.09 PAH b: 0.15 Chung et al., 2011 smoked sausages processed products Bacon processed products Serbia 1.09 0.24 PAH c: 21.3 PAH c: 4.4 Djinovic et al., 2008 smoked beef ham smoked cajna smoked beef ham smoked cajna sausage sausage Spain 3.21 ± 0.12 0.38±0.08 Ledesma et al., 2015a smoked chorizo smoked chorizo Sweden 36.9 n.d PAH4:209 PAH4: n.d Wretling et al., 2010 smoked ham various smoked smoked ham various products

Consideraciones teóricas Table 7. (continued). PAH ( •g/kg) World Smoked Food BaP PAH Reference Maximun Minimiun Maximun Minimun Smoked fish from Africa (Republic of Ghana) 73.78 ± 11.44 (max) 3.36 ± 4.52 (mean) PAH d: 1461.79 PAH d: 510.59 Essumang et al., 2012 52.00 ± 18.88 •g/kg (mean) smoked herrings smoked herrings smoked herrings smoked herrings Denmark 11.4 <0.2 PAH e: 78 PAH e: 1.1 Duedahl-Olesen et al., 2010 smoked cod roe Various smoked cod roe smoked trout Iran 7.56 35 <2 PAH g: 110 ± 89 PAH g: 26 ± 17 Forsberg et al., 2012 smoked salmon smoked salmon smoked salmon smoked salmon Smoked cheese Circassia/Turkey 1.52 n.d PAH h :3.51 PAH h :0.11 Gul et al., 2015 smoked Circassian cheese smoked Circassian smoked Circassian smoked Circassia n cheese cheese cheese Italy 417.8 n.d PAH4: 1892.5 PAH4: n.d Esposito et al., 2015 rind of mozzarella smoked smoked mozzarella rind of mozzarella with corrugated cardboard smoked with corrugated cardboard Spain 0.52 ± 0.03 n.d PAH i: 1,037.23 PAH i: 36.31 Guillén & Sopelana, 2004 smoked sheep cheese rind smoked cheese Spain 0.51 n.d PAH i: 739.55 PAH i: 29.21 Guillén et al., 2007 smoked Palmero cheese rind smoked PalmeroTable cheese 7. (continued). smoked Palmero cheese smoked Palmero cheese

Capítulo 2 Table 7. (continued). PAH ( •g/kg) World Smoked Food BaP PAH Reference Maximun Minimiun Maximun Minimun Smoked beverage Tea from different n.d n.d PAH4: 2.7 PAH4: <0.4 Pincemaille, Schummer, manufactureres smoked black tea smoked black tea smoked black tea smoked black tea Heinen, & Moris, 2014 Tea from different 237 0.8 black tea and PAH j: 1,997.9 PAH j: 13.8 Ziegenhals, Jira, & Speer, manufactureres mate tea he rbal and fruit tea mate tea herbal and fruit tea 2008 Tea from different 73.2 ± 8.5 n.d PAH k: 628.8 PAH k: 55.4 Ishizaki, Saito, Hanioka, manufacturers black tea (Kayuaro) other types of tea (smoked) Oolong black tea (Darjeeling) Narimatsu, & Kataoka, 2010 tea Tea from different 460 n.d PAH4: 1700 PAH4: n.d Schulz, Fritz & Ruthenschrör, manufacturers Lapsang souchong tea other types of tea Lapsang souchong other types of tea 2015 tea Tea from different 380 n.d PAH4: 1400 PAH4: n.d Schulz et al., 2015 manufacturers mate tea other herbal and mate tea other herbal & fruit tea fruit tea a: PAH: BaA, BbF, BjF, BkF, BghiP, BaP, Ch, DBahA, DBaeP, DBalP, IP, 5MeCh. b: PAH:! PAH: CHR, BbF, BkF, BaP, DBahA, BghiP, IP c: PAH: BcL, BaA, CPP, CHR, 5MC, BbF, BjF, BkF, BaP, BgP, DhA, IcP, DeP, DhP, DiP, DlP d: PAH: Na,Ap,Ac,F, A, Pa, Fl,P,BaA,BkF,IP, BbF,Ch, BaP, BghiP, DBahA e: SCF: BaA, CHR, CPP, 5MeCHR, BbF, BjF, BkF, BaP, IND, DBahA, BghiP, DBalP, DBaeP, DBaiP, DBahP. f: PAH8: BaA,Ch,BbF, BkF, BaP,DBahA, BghiP, IP g:! PAH: BaP and relative potency factor adjusted of Fl, BbF, BaA, Ch, BkF h: PAH5: BaA, BkF, BghiP, BaP, DBahA i:! PAH:!sum!of!more!than!50PAH j:! PAH:!BcL!BaA,!CHR,!TP,!CPP,!5MC,!BbF,!BkF,!BjF,!BaP,!IcP,!DhA,!BgP,!DlP,!DeP,!DiP,!DhP k:! PAH:!Na,!Ac,!Fl,!Pa,!A,!Fl,!P,!BaA,!Chr,!BbF,!BkF,!BaP,!DaHA,!BghiP,!IP *The Confederated Tribes of the Umatilla Indian Reservation (CTUIR), Columbia River Basin Traditional Native American.

Consideraciones teóricas

As table 7 shows, PAH have been found in all types of smoked food. Particular attention has traditionally been paid to smoked meat products, because the highest levels of total PAH have been detected in these food (Gomaa, Gray, Rabie, Lopez-Bote, & Booren, 1993; Karl and Leinemann, 1996; Larsson, Pyysalo, & Sauri, 1988; Martorell et al., 2010) and because they are the major contributors to PAH dietary intake in some developed locations (Martorell et al., 2010). However low values of below 1 •g/kg have recently been reported in smoked meat products from developed countries (Jira, Ziegenhals, & Speer, 2006; Purcaro et al., 2009; Roseiro et al., 2012; Santos et al., 2011). The legislation and some modern smoking techniques, such as liquid smoke or indirect smoking methods help to reduce the PAH content of smoked food in these countries.

From a global perspective, the Codex Alimentarius states that the major contributors to PAH intake are cereals and cereal products (owing to high consumption in diets) and vegetable fats and oils (due to higher concentrations of PAH in this food group) (Codex Alimentarius CAC/RCP 68/2009). As table 7 shows, special attention should be paid in the developing countries and some ancient societies, because of the high amounts of PAH that have being reported in smoked food from these locations, such as 73.78 ± 11.44 •g/kg of BaP in smoked herrings in Africa (Essumang, Dodoo, & Adjei, 2012), o r more than 35 •g/kg in smoked salmon in traditional native American tribes (Forsberg et al., 2012). The National Training Centre for Fisheries and Aquaculture Technicians (CNFTPA) of Senegal in collaboration with FAO have designed and developed the FTT-Thiaroye technique. This technique and its diffusion help to minimize the PAH content of the smoked food in the developing countries (FAO-Thiaroye, 2015).

As table 10 shows, a high level of PAH has been found in some smoked teas, such as 237, 73.2 ± 8.5, a nd 460 •g/kg of BaP in mate (Ziegenhals et al., 2008), Kayuaro black tea (Ishizaki et al., 2010), and Lapsang souchong tea (Schulz et al., 2014), respectively. Smoked food are not the only contributors to PAH in the diet, PAH have been detected in roasted and toasted food, like coffee (Houessou, Delteil, & Camel, 2006), toasted bread (Rey-Salgueiro, García-Falcón, Martínez- Carballo, & Simal-Gándara, 2008), and non processed food, such as vegetables, eggs, oils, shellfish, fruits or milk (Martorell et al., 2010), probably because of environmental contamination (CAC/RCP 68/2009; Rodríguez-Acuña, Pérez-Camino, Cert, & Moreda, 2008).

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5.1.5 Prevention

PAH contamination in smoked food can be prevented, maintaining the beneficial effects of smoking. The “Code of practice for the reduction of contamination of food with (PAH) from smoking and direct drying processes” (CAC/RCP 68/2009) provides the variables that need to be controlled to minimize the PAH contamination of smoked food. The variables have been studied by several researchers in different smoked food. They include the kind of fuel, studied in smoked meat (García-Falcon & Simal-Gándara, 2005; Hitzel et al., 2013; Pöhlmann et al., 2012; Stumpe- V!ksna et al., 2008 ) and smoked cheese (Conde, Ayala, Afonso, & González, 2005; Esposito et al., 2015; Guillén et al., 2007), smoking method, studied in smoked meat (Pöhlmann et al., 2012, 2013a; Škaljac et al., 2014 ), smoked fish (Duedahl-Olesen, Christensen, Højgård, Granby, & Timm- Heinrich, 2010; Hattula, Elfving, Mroueh, & Luoma, 2001; Karl & Leinemann, 1996; Varlet et al., 2007; Varlet, Serot, Monteau, Le Bizec, & Prost, 2007), and smoked cheese (Aydinol & Ozcan, 2013; Esposito et al., 2015), smoking duration, studied in smoked meat (Djinovic et al., 2008; Ledesma et al., 2014), and fish food (Essumang et al., 2013) fat content, studied in smoked meat products (Gomes et al., 2013; Pöhlmann et al., 2013b) and its evolution during processing (Ledesma et al., 2015b), distance and position between the food and the heat source, studied in smoked meat products (Pöhlmann et al., 2013b) and smoked cheeses (Guillén et al., 2011), cleanliness and maintenance of equipment, and the design of the smoking chamber and the equipment (Gomes et al., 2013; Pöhlmann et al., 2013a). The casing type is also an important and recently studied variable (García-Falcon & Simal-Gándara, 2005; Gomes et al., 2013; Ledesma et al. , 2015b; Pöhlmann et al., 2013b; Škaljac et al., 2014).

Among all the variables recommended by CAC/RCP 68/2009, three seem to have a greater influence on preventing PAH contamination: the temperature of smoke generation, the type of casing and the smoking method (direct or indirect). Temperature is the most important variable to be considered (Pöhlmann et al., 2012). A smoke generation temperature below 600°C should be applied to prevent the formation of PAH (Pöhlmann et al., 2012). The use of synthetic instead of natural casings prevents PAH from penetrating inside smoked food (Garcia-Falcón & Simal-Gándara, 2005; Gomes et al., 2013; Ledesma et al., 2015b; Skaljac et al., 2014). Indirect smoking systems (e.g., friction or liquid smokes) can highly reduce the PAH content of food (Hattula et al., 2001; Pöhlmann et al., 2013a; Varlet et al., 2007). On the other hand, the control of other variables such as wood (never gasoline, treated woods or wasted oil), smoking time, fat

Consideraciones teóricas

content, some types of pretreatment such as marinating (Farhadian, Jinap, Faridah, & Zaidul, 2012), microwave preheating, and aluminum wrapping (Farhadian, Jinap, Hanifah, & Zaidul, 2011), and the addition of onion and garlic as meat additives (Janoszka, 2011), can help to reduce the final PAH content of grilled and smoked food.

5.2 Other toxic compounds

During the food smoking process other toxic compounds are produced, such as formaldehyde, N-Nitrosamines, heterocyclic aromatic amines, •-carbolines and • -carbolines. These compounds have toxic effects on smoked food and could also contaminate the ambient air of the food factories.

5.2.1 Formaldehyde

Formaldehyde (CH 2O) is an important compound for the global economy, widely used in construction, wood processing, furniture, textiles, carpeting, and in the chemical industry (Tang et al., 2009). Formaldehyde is naturally present in the environment, some food and in the human body (IARC, 2006). However, some industrial processes and food processing methods such as smoking increase the formaldehyde content up to contaminant levels (IARC, 2006). Formaldehyde can be found in several non smoked food, including fruit, vegetables, milk, beverages or eggs because of environmental contamination of raw materials (Feron et al., 1991; IARC, 2006), but its concentration could be increased during cooking processes at high temperatures and because of the exposition to smoke from wood combustion (Herrmann, Granby, & Duedahl-Olesen, 2015). Formaldehyde has bacteriostatic and bactericidal effects on smoked food (Rørvik, 2000), that help to prolong the products shelf-life (Goulas & Kontominas, 2005). However, it is carcinogenic to humans (IARC, 2006; Vincent et al., 2005). Formaldehyde is added inappropriately in some food because of its preservative effect (Tang et al., 2009; Wang, Cui, & Fang, 2007; Zhao & Zhang, 2009). Formaldehyde as additive in food, in animal feeding, and as a contaminant in fermented alcoholic beverages is regulated by the U.S. Food and Drug Administration (FDA) (EPA, 1989, 2007), the European Union (EU, 2004/C 50/01) and China (MOH, 2005), respectively. A few research works have been focused on the formaldehyde contamination of smoked food. A high concentration of formaldehyde, up to 125 mg/kg wet weight, has been found in smoked meat products (Zhu, Peng, Wang, Wang, & Rui, 2012). A lower concentration of formaldehyde has been found in smoked fish, such as 855 ± 56

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•g/kg in smoked salmon (Gosetti et al., 2011). The effect and toxic contamination of formaldehyde in smoked fish (Varlet, Prost, & Serot, 2007) and other smoked food should be further investigated.

5.2.2 N-Nitrosamines

N-Nitrosamines (NAs) are a group of compounds, of which many have been classified as genotoxic and probable human carcinogens (IARC, 1978). Several factors affect the NAs contamination of food. The most known factor is the addition of nitrite (E 249 –E 250) or nitrate (E 251 –E 252) during salting, and smoking processes (Herrmann, Duedahl- Olesen, Christensen, Olesen, & Granby, 2015). There is a positive though not necessarily linear correlation, between the amount of nitrite added and the amount of NAs formed (Drabik- Markiewicz et al., 2009, Drabik-Markiewicz et al., 2011; Yurchenko & Mölder, 2007). According to the European Food Safety Authority (EFSA) opinion, expressed on 26 November 2003, the maximun levels of nitrites and nitrates allowed to add to food products have been reduced by the Directive 2006/52/EC, in order to keep the level of nitrosamines as low as possible (EC, 2006/52). Other factors have an important, even more significant, role in the NA content of the final products, such as some precursors added via wood smoke, spices or other ingredients (Herrmann et al., 2015), and the heat applied during drying and smoking processes (Herrmann, Duedahl-Olesen, & Granby, 2015). Some nitrosamines, such as N-nitroso- thiazolidine-4-carboxylic acid (NTCA) and N-nitroso-2-methyl-thiazolidine 4-carboxylic acid (NMTCA) can be found in smoke (Massey et al., 1991) and its production increases by increasing the temperature during the smoking process (Herrmann et al., 2015). Several works have been focused in the determination of NAs in smoked food, such as smoked meat products (Yurchenko, & Mölder, 2007), smoked fish (Herrmann et al., 2015; Yurchenko, & Mölder, 2006), smoked beverages like Rauchbier beer (Mangino, 1981), even in smoked oysters (Lijinsky, 1999). A high concentration of various nitrosamines has been found in smoked food, such as 32 •g/kg of dimethylamine in smoked pickled fish, 10 •g/kg of pyrrolidine in smoked meat, 9 •g/kg of piperidine in spiced smoked meat, 2100 and 167 •g/kg of proline in smoked pork and smoked oyster, respectively (Lijinsky, 1999).

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5.2.3 Heterocyclic aromatic amines

Heterocyclic aromatic amines (HAAs) are an important group of food mutagens in the Ames/salmonella assay. These compounds are potential dietary carcinogens in rodents and primates (Ka-Wing et al., 2006). The factors affecting the content of HAAs in food have recently been reviewed (Alaejos & Afonso, 2011). HAAs are formed during heating processes, thereby including food smoking process (Szterk & Waszkiewicz-Robak, 2014). Its formation mainly depends on the process temperature (Alaejos & Afonso, 2011), but other factors, such as cooking time and method (including smoking), concentration of precursors, presence of enhancers or inhibitors, amount of lipids or water and pH have also an important role (Bordas, Moyano, Puignou, & Galceran, 2004; Juhee & Ingolf, 2005; Ristic, Cichna, & Sontag, 2004; Skog, Eneroth, & Svanberg, 2003). Several studies have been focused on the formation and prevention of HAAs in grilled and roasted and grilled food (Alaejos & Afonso, 2011). A few studies are focused on the HAAs content of smoked food, such as smoked pork rib (Knize et al., 1998), smoked fish, such as salmon and flounder (Johansson & Jägerstad, 1994) and smoked cheeses, like Provola (Naccari et al., 2009). Like PAH (Guillén & Sopelana, 2004), highest levels of HAAs were found in the cheese rind, the part in direct contact with smoke during the smoking processes, and then could migrate via the fats to reach the interior part of the smoked cheese, where less concentration was found (Naccari et al., 2009). Some authors have studied the role of smoke flavourings in HAAs production and concentration (Solvakov et al., 1999; Stavric et al., 1997). Traditional smoking techniques contribute to the final HAAs content of the food (Naccari et al., 2009), while the use of filters or liquid smoke flavourings reduces the amount of HAAs in the final product by almost 100% (Naccari et al., 2009; Simon, De la Calle, Palme, Meier, & Anklam, 2005). Liquid smoke seems to be a safe technique to prevent HAAs contamination of smoked food. In fact, the composition of smoke flavor is regulated by the European Parliament and Council Regulation EC No 2065/2003, and no reference to HAs is mentioned (EC, 2065/2003; Naccari et al., 2009).

5.2.4 • -carbolines

•-carbolines are a group of compounds that lead to toxic activity on food. The genotoxic potential and co-mutagenic properties these compounds have been reviewed (De Meester, 1995). S ome • -carbolines interact with various macromolecules and enzymatic systems and may modify and increase the genotoxic and toxic consequences of other toxic compounds cited here, such as PAH or n-nitrosamines (De Meester, 1995). The smoking process can increase the

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concentration of these compounds in foodstuff (Papavergou & Clifford, 1992). Smoked products appear to be one of the food stuffs with the highest amount of some • -carbolines, such as tetrahydro-b-carboline (Papavergou & Herraiz, 2003). Some studies (Papavergou & Herraiz, 2003; Sen, Seaman, Lau, Weber & Lewis, 1995) have been focused on the presence of some • - carbolines in smoked food . The main • -carboline in smoked food is 1,2,3,4-tetrahydro- •- carboline-3-carboxylic acid, which concentration ranges between 0.03 –12.2 !g/g, 0.07–6.06 !g/g, and 0.01 –14.8 !g/g in smoked fish, smoked cheeses and smoked sausages and meats, respectively (Papavergou & Herraiz, 2003). Like PAH (Guillén & Sopelana, 2004) and HAAs (Naccari et al., 2009) the highest levels of this contaminant were found in the external part or the products, which is in direct contact with smoke during the smoking processes (Papavergou & Herraiz, 2003).

6. Conclusions

Smoked food are one of the most ancient preserved food and have had an important role on humans diet and economical markets. The main smoked food are smoked meat, fish and cheese, but smoking is also applied to some beverages and spices. Nowadays the smoking technique has been improved and can be replaced, in developed countries, by more controlled and optimized indirect smoking methods, such as liquid smoke and smoke produced by friction generation, and other methodologies for fresh food preservation. Several compounds are produced during smoking, conferring desirable and undesirable effects on the smoked food. The desirable effects are food preservation, which is still the main aimed effect in developing countries, and the improvement of food organoleptic profile, colour, texture and flavor, which is nowadays the main aimed effect in developed countries. The main undesirable effect of smoking is the contamination of food by toxic and carcinogenic compounds, such as PAH, n-nitrosamines, heterocyclic aromatic amines and •-carbolines. These compounds are produced in the traditional smoking processes and are mainly accumulated in the surface of the food, such as the meat product casing and the cheese rind. Thereby the external surface of smoked food should be removed before the consumption. The application of the modern smoking technologies and the direct smoking process in a controlled way, accordingly to Codex Alimentarius (CAC/RCP 68/2009), let maintain the desirable effects and reduce or prevent the contamination of smoked food by toxic compounds. This is especially important in developing countries, where direct smoking is still applied in the traditional way as the main food preservation method.

Consideraciones teóricas

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EPA (1989). US Environmental Protection Agency (EPA). Integrated Risk Information System on Formaldehyde, National Center for Environmental Assessment, Office of Research and Development, Washington, DC, 1999. Retrieved April 8, 2015 from: http://www.epa.gov/iris/subst/0419.htm

Consideraciones teóricas

EPA (2007). US Environmental Protection Agency (EPA). Formaldehyde TEACH Chemical Summary U.S. EPA, Toxicity and Exposure Assessment for Children’s Health. Retrieved April 8, 2015 from: http://www.epa.gov/teach/chem_summ/Formaldehyde_summary.pdf

EPA (2013). US Environmental Protection Agency (EPA). National Primary Drinking Water Regulations (EPA 816-F-09-0004, May 2009). Retrieved October 10, 2014, from: http://water.epa.gov/drink/contaminants/index.cfm#List

EU, 2004/C 50/01. List of the authorised additives in feedingstuffs published in application of Article 9t (b) of Council Directive 70/524/EEC concerning additives in feedingstuffs. Retrieved April 8, 2015 from: http://eur- lex.europa.eu/LexUriServ/LexUriServ.do?uri=OJ:C:2004:050:0001:0144:EN:PDF

EU, No 835/2011. European Union Commission Regulation (EU) No 835/ 2011 of 19 August 2011 amending Regulation (EC) No 1881/2006 as regards maximum levels for polycyclic aromatic hydrocarbons in foodstuffs. Retrieved September 10, 2014 from: http://eurlex.europa.eu/LexUriServ/LexUriServ.do?uri=OJ:L:2011:215:0004:0008:EN:PDF

FAO (2008). Food and Agriculture Organization of United Nations (FAO). International exports of fishery commodities by the Harmonized System and FAO ISSCFC (2008). Retrieved April 5, 2015 from: ftp://ftp.fao.org/fi/stat/summary/summ_09/a4ybc.pdf

FAO (2013). Food and Agriculture Organization of United Nations (FAO). Agriculture and Consumer Protection Department. Animal production and health. Meat & Meat Products. Retrieved August 10, 2014 from: http://www.fao.org/ag/againfo/themes/en/meat/home.html

FAO (2014a). Food and Agriculture Organization of United Nations (FAO). Agriculture and Consumer Protection Department. FAO Corporate Document Repository. “World Agriculture: Towards 2015/2030. An FAO perspective...”. Retrieved August 10, 2014 from: http://www.fao.org/docrep/005/y4252e/y4252e05b.htm

FAO (2014b). Food and Agriculture Organization of United Nations (FAO). The State of World Fisheries and Aquaculture. (“El estado mundial de la pesca y la acuicultura”). Retrieved April 5, 2015 from: http://www.fao.org/fishery/en

Capítulo 2

FAO (2015). Food and Agriculture Organization of United Nations (FAO). Smallholder tea producers benefit from harmonized safety standards. Retrieved April 5, 2015 from: http://www.fao.org/in-action/smallholder-tea-producers-benefit-from-harmonize-safety- standards/en/

FAO-Thiaroye (2015). National Training Centre for Fish and Aquaculture Technicians Thiaroye (Thiaroye)-Food and Agriculture Organization of United Nations (FAO). Guide for developing and using The FAO-Thiaroye Processing Technique (FTT-Thiaroye). Retrieved April 5, 2015 from: http://www.fao.org/3/a-i4174e.pdf and http://www.fao.org/news/audio-video/detail- video/en/c/10566/?uid=10566

Fessmann, G. (1972). Smoke flavouring. Process for the production of a smoking fluid for smoking foodstuffs. Patent US3634108. Retrieved April 5, 2015 from: http://worldwide.espacenet.com/publicationDetails/biblio?CC=US&NR=3634108A&KC=A&FT=D

IARC (1978). (International Agency for Research on Cancer). Monograghs on the evaluation of carcinogenic risks to humans. Some N-Nitroso compounds. Retrieved April 8, 2015 from: http://monographs.iarc.fr/ENG/Monographs/vol1-42/mono17.pdf

IARC (2006a). (International Agency for Research on Cancer). Monograghs on the evaluation of carcinogenic risks to humans. Formaldehyde, 2-butoxyethanol and 1-tert- buxy-2-propanol. IARC, Lyon, France, 2006. Retrieved April 8, 2015 from: http://monographs.iarc.fr/ENG/Monographs/vol88/mono88.pdf

Miler, K., & Kozlowski, Z. (1969). Method of producing a smoke preparation. Patent US3445248. Retrieved April 5, 2015 from: http://worldwide.espacenet.com/publicationDetails/biblio?CC=US&NR=3445248A&KC=A&FT=D

Consideraciones teóricas

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SCF (2002). Scientific Committee on Food (SCF). Opinion of the Scientific Committee on Food on the risks to human health of Polycyclic Aromatic Hydrocarbons in food. Retrieved September 10, 2014 from: http://ec.europa.eu/food/fs/sc/scf/out153_en.pdf

USDA/HHS (2010).United States Department of Agriculture (USDA) and United States Department of Health and Human Services (USHHS). Dietary Guidelines for Americans 2010. Retrieved October 9, 2014 from: http://www.cnpp.usda.gov/sites/default/files/dietary_guidelines_for_americans/PolicyDoc.pdf

WHO (2006). World Health Organization (WHO). Genova 2006. The Joint FAO/WHO Expert Committee on Food Additives, JECFA. Evaluation of certain food contaminants. Sixty-fourth report of the Joint FAO/WHO Expert Committee on Food Additives. WHO Technical Report Series, No 930, 61-79. Retrieved August 15, 2014 from: http://whqlibdoc.who.int/trs/WHO_TRS_930_eng.pdf

WHO/FAO (2003). World Health Organisation (WHO)/ Food and Agriculture Organization of United Nations (FAO). Diet, nutrition and the prevention of chronic diseases. WHO Technical Report Series 916. Retrieved October 9, 2014 from: http://whqlibdoc.who.int/trs/who_trs_916.pdf

•••••••••••••••••••••••••••••••••••••••••••••••••••Materiales••y•métodos• • • • • • • • Materiales y métodos

3.•MATERIALES•Y•MÉTODOS•

3.13 .1 OBTENCIÓN DE MUESTRAS

3.1.1 TRIPAS

Las tripas estudiadas en esta tesis han sido de dos tipos, natural (Viuda de Inocencio Fernández, España), y artificial (Fibrán, España). Todas fueron facilitadas por El Hórreo Healthy Food S.L (Noreña, España).

La tripa natural utilizada, procede de intestino delgado de cerdo, tiene un calibre dado en el rango 28/30 cm y se adquirieron presentadas en madejas. Estas tripas se conservan en refrigeración, antes de su uso. Las tripas naturales más comunes en el mercado son de origen porcino, vacuno, ovino, caprino y equino. Se comercializan en barriles, cubos de distintas capacidades, en bolsas (saladas o en salmuera), entubadas y en madejas. En el mercado se presentan tripas de distintos calibres, desde 26/28 cm hasta 44/46 cm, en intestino delgado de cerdo, y otros formatos procedentes del intestino grueso del cerdo, incluyendo las tripas de cerdo vuelto, semirizada, semicular, cular, ciegos y vejigas (Viuda de Inocencio Fernández S.A, 2015).

La tripa artificial utilizada, se clasifica en la categoría “ colágeno ”, y tiene un calibre de 26/28 cm. La ficha técnica de este producto, facilitada por el fabricante, clasifica el producto en la tipología S1. Su composición es la siguiente: 74,7% colágeno, 18,0% glicerina, 2,0% grasas, 0,3% cenizas y 5,0% agua. Entre sus especificaciones físicas se encuentran un grosor de 0,04-0,09 mm y una elasticidad del 15-20%. Estas tripas se almacenan en un lugar fresco y seco, protegido de la luz solar, antes de su refrigeración. Existen otras tipologías de tripas artificiales tanto comestibles como no comestibles, de celulosa, de fibra, textiles, de lino, de poliéster, cloruro de polivinilideno (PVDC), poliamida (nylon), y multicapas (nylon, poli-olefina, y otros polímeros), cuya clasificación es revisada por Savic (2012).

Capítulo 3

3.1.2 CHORIZO

3.1.2.1 Muestras de chorizo elaboradas

Todas las muestras de chorizo elaborado específicamente para este trabajo fueron fabricadas en la empresa El Hórreo Healthy Food S.L. Las muestras se pueden clasificar en 2 tipos, chorizo embutido en tripa natural (ChN) y chorizo embutido en tripa sintética (ChS). Las muestras de chorizo fueron tomadas en las instalaciones de la empresa durante días productivos, con el objetivo de reproducir las condiciones reales de fabricación. La cantidad de chorizo fabricada para cada estudio fue de 50kg, cantidad considerada como una producción a escala industrial.

Las proporciones de ingredientes seleccionados para la elaboración de los chorizos estudiados fueron: magro de cerdo (46,8%), panceta de cerdo (46,8%), sal (1,8%), ajo (1%), pimentón dulce (2%), y especias y aditivos (1,6%). La mezcla de especias y aditivos está formada por ingredientes para garantizar la estabilidad microbiológica y el control de calidad de los chorizos, como dextrina, dextrosa, citrato sódico (E-331iii), polifosfatos de sodio (E-451i), ascorbato sódico (E-301), nitrito sódico (E-250), y el colorante (E-120).

El magro de cerdo, y la panceta de cerdo, se picaron por separado en una picadora industrial (Taffo, España), y posteriormente se mezclaron, junto al resto de ingredientes, en una amasadora de vacío (Tec Maq, España).

a. Picado b. Amasado Figura 3.1. Picado (a) y amasado (b) de los ingredientes para la fabricación de muestras de chorizo.

Del resultado del picado y mezclado de los ingredientes, se obtiene una masa, que se deja reposar durante 1 día. Este tiempo es necesario para permitir la maceración de la mezcla y que tengan lugar los primeros procesos de fermentación del producto. Una vez pasado este tiempo, la masa pasa a la embutidora (Handtmann, Alemania), donde es embutida en la tripa

Materiales y métodos

estudiada, natural o artificial, previamente remojada en agua con sal. Las siguientes imágenes muestran el proceso de embutido.

Figura 3.2. Selección y remojado de la tripa.

Figura 3.3. Proceso de embutido de la masa en la tripa.

Capítulo 3

Por cada 50 kg de producción se elaboraron unas 100 riestras de chorizos, conteniendo cada riestra 4 unidades de chorizo, las cuales fueron colgadas en barras y situadas en estanterías, tal y como muestra la siguiente figura.

Figura 3.4. Colocación de chorizo en estantería para llevar a la cámara de ahumado.

Una vez colgados los chorizos, se situó la estantería en la cámara de ahumado, la cual se encuentra a una distancia de 10 m respecto a la fuente de humo. Si una cantidad de chorizo menor es introducida en la cámara de ahumado, puede ser expuesta a una cantidad excesiva de humo, por lo que las muestras se realizaron con cantidades industriales de chorizo (50kg por estantería). Para el ahumado directo de las muestras se utilizaron maderas de roble y castaño, especificándose las proporciones es en cada trabajo. Las condiciones de humedad y temperatura de las muestras ahumadas al modo directo están definidas por las condiciones meteorológicas y se indican en cada estudio. El tiempo de exposición de los chorizos varía en función del estudio, desde los 0 a los 11 días de ahumado.

La siguiente figura esquematiza la cámara de ahumado tradicional utilizada.

Materiales y métodos

Figura 3.5. Cámara de ahumado directo industrial (tradicional) de El Hórreo Healthy Food S.L.

Capítulo 3

La siguiente figura resume el flujo del proceso de fabricación de chorizo.

FAMILIA 1: EMBUTIDOS CURADOS / AHUMADOS

RECEPCIÓN RECEPCIÓN RECEPCIÓN AGUA RECEPCIÓN RECEPCIÓN ENVASES / MATERIAS ESPECIAS / TRIPA TRIPA EMBALAJES PRIMAS ADITIVOS NATURAL ARTIFICIAL PCC1

ALMACENAMIENTO ALMACENAMIENTO ALMACENAMIENTO ALMACENAM. ALMACENAM. ENVASES / ESPECIAS / MATERIAS PRIMAS TRIPAS TRIPAS EMBALAJES ADITIVOS

DESCONGELACIÓN

LOMO PREPARACIÓN LAVADO PICADO HIDRATACIÓN EMBUCHADO MEZCLA TRIPAS

AMASADO

MACERACIÓN

EMBUTICIÓN

ATADO

SECADO DFL.004

AHUMADO

ENVASADO

IDENTIFICACIÓN /ETIQUETADO

EMBALAJE

EXPEDICIÓN

Figura 3.6. Diagrama de flujo del proceso de fabricación de muestras de chorizo.

Materiales y métodos

3.1.2.2 Chorizos comerciales

Todas las muestras estudiadas en este trabajo, en cuya descripción no se indica haber sido elaboradas en las instalaciones de El Hórreo Healthy Food S.L son muestras de chorizo adquiridas en establecimientos comerciales. Entre esta tipología de muestras en total se estudiaron chorizos ahumados elaborados por 16 diferentes empresas de productos cárnicos del Principado de Asturias.

3.1.3 ACEITE

El aceite utilizado en este trabajo fue aceite de oliva extra virgen (EVOO) (Carbonell, España), adquirido en establecimientos comerciales. Los hidrocarburos aromáticos policíclicos son compuestos lipofílicos (Vázquez Troche, García Falcón, González Amigo, Lage Yusty, & Simal Lozano, 2000), que se disuelven en las grasas en estado líquido de manera uniforme. Para evaluar las diferencias de penetración de BaP a través de los distintos tipos de tripa, se diseñó un nuevo sistema ideal de dilución, formado por aceite contenido en los distintos tipos de tripa.

En primer lugar, se tomaron 40 cm de ambas tripas, natural y artificial y se remojaron en agua con sal durante 30 minutos, y seguidamente se llenaron las tripas con 400 mL de EVOO, tal y como muestra la figura 3.7.

a b c

Figura 3.7. Selección y medida (a), remojo (b) y llenado (c) de las tripas.

Capítulo 3

Se guardaron en la botella original 40 mL de aceite sin procesar, en la oscuridad, para su análisis (aceite sin ahumar). Las muestras se colgaron separadas 40 cm una de la otra en la misma barra de una estantería, tal y como muestra la figura 3.8.

Figura 3.8. Sistema de aceite en distintos tipos de tripa, colgado en la estantería.

La estantería se emplazó en la cámara de ahumado, de manera que ambos sistemas se encontraron en la misma posición y localización de la cámara de ahumado, a la misma distancia de la fuente generadora de humo, 10 m, y se ahumaron durante 7 días empleando una mezcla de roble (90%) y castaño (10%).

Una vez ahumadas, las muestras se colgaron en una barra situada dentro de una caja de cartón y fueron transportadas hasta el laboratorio de análisis, tal y como muestra la figura 3.9.

Figura 3.9. Transporte de sistemas de aceite en distintos tipos de tripa.

Materiales y métodos

El contenido de aceite de las muestras se guardó en matraces individuales de cristal, a 5ºC en la oscuridad tal y como muestra la figura 3.10.

Finalmente, se analizaron 15 muestras, 3 por cada uno de los siguientes productos: aceite original (sin ahumar), aceite ahumado embutido en la tripa natural, aceite ahumado embutido en la tripa sintética, tripa natural ahumada y tripa sintética ahumada.

Figura 3.10. Preparación de sistema de aceite en tripa natural y colágeno, y toma de muestras.

3.2 DETERMINACIÓN DE BENZO(A)PIRENO

3.2.1 REACTIVOS Y PATRONES

La solución de patrón analítico Benzo(a)pireno (BaP) fue obtenida en Sigma-Aldrich (St. Gallen, Alemania), con una concentración de 100 µg/mL en ciclohexano y un volumen total de 2 mL. La solución del patrón interno Benzo(a)pireno (BaP d12, 98%), fue obtenida de Cambridge Isotope Laboratories (Andover, MA, EEUU), a una concentración de 200 µg/mL en isooctano y un volumen de 1,2 mL. Todos los disolventes para la preparación de muestras, hexano (ref. 32293), diclorometano (ref. 66740), ciclohexano (ref. 2893) e isooctano (2, 2, 4-Trimetilpentano) (ref. 59050), fueron suministrados por Sigma- Aldrich con una pureza de grado ACS• 99% (GC).

Los cartuchos de extracción de fase sólida (SPE) fueron suministrados por Varian (Palo Alto, California, Estados Unidos) y Agilent Technologies (Santa Clara, California, Estados Unidos).

Capítulo 3

Se emplearon cartuchos Mega Bond Elut, los cuales contienen fase adsorbente de sílice, con un tamaño de partícula de 40nm, una masa de 5g y un volumen de 20 mL. Se utilizaron matraces y viales de color ámbar (para proteger a los compuestos a determinar de la descomposición debida a la luz. Se utilizaron específicamente matraces de cristal ámbar de 15 mL (21 x 70 mm) adquiridos en Sigma-Aldrich para la recolección de los HAP durante la etapa SPE.

3.2.2 PRETRATAMIENTO DE MUESTRAS DE CHORIZO

Para llevar a cabo los análisis de contenido de BaP en las muestras, es necesario realizar un proceso de pretratamiento de las muestras para la extracción del analito de interés. En la siguiente figura 3.11, se esquematizan los pasos involucrados en los métodos de pretratamiento de muestras probados en el laboratorio. Seguidamente se detallan los pasos de la vía analítica finalmente seleccionada y utilizada en todos los experimentos de este trabajo, la cual consiste en la combinación de sonicación y extracción en fase sólida (SPE) (vía de la derecha).

Figura 3.11. Pretratamiento.

Materiales y métodos

3.2.2.1 Selección de la muestra y alícuotas a analizar

En primer lugar, se caracterizaron las muestras, tomando medidas de su longitud, diámetro e inspección visual. Seguidamente, se seleccionaron las alícuotas de las muestras a analizar. El procedimiento de pretratamiento de las muestras se realizó por triplicado (3 alícuotas de cada muestra) en todos los estudios. En función del tipo de estudio los métodos de toma de muestras utilizados se pueden clasificar en 2 tipos:

a) Toma de muestras del producto completo homogeneizado (sin la tripa).

b) Separación de producto en diferentes profundidades, y toma de muestras.

Figura 3.12. Selección y cuarteamiento de muestras.

3.2.2.2 Picado

Una vez seleccionadas las muestras, se picaron y homogeneizaron con ayuda de una picadora de carne (Minirobot D81 de Moulinex, France). También se utilizó en un molinillo de café (Moulinex, France) para pulverizar muestras, tal y como sugieren Purcaro, Moret, y Conte (2009).

3.2.2.3 Liofilización

Se tomaron 3 alícuotas de 20g de chorizo y se colocaron en platos individuales del liofilizador (Telstar-Cryodos, España), cuidadosamente cubiertos con papel de aluminio y se pesaron antes del proceso de liofilización. Cada muestra fue congelada a -80ºC durante 4 horas y liofilizada en un liofilizador durante 1 día. Finalmente se pesaron los platos, conteniendo las muestras secas.

Capítulo 3

Figura 3.13. Equipo de liofilización.

3.2.2.4 Sonicación

Se llevaron 2 g de chorizo liofilizado de cada plato a matraces de base ancha, de 250 mL. La cantidad sobrante de chorizo se guardó en un congelador. Seguidamente, se añadieron 20 mL de n-hexano y 200 µL de la solución estándar interno de benzo(a)pireno-d12 con una concentración de 100 µg/L (habiendo hecho las diluciones de la disolución madre previamente).

Estas disoluciones se mantuvieron bajo refrigeración durante 3 días para permitir que el patrón interno deuterado se adaptase a la matriz de la muestra. Este es el momento apropiado para añadir el estándar interno (y adiciones estándar para determinar la recuperación) pues es el primer momento en el que ambos compuestos se encuentran en las mismas condiciones y en el mismo medio de dilución (Purcaro et al., 2009; Stumpe-V•ksna, Bartkevics, Kukare, & Morozovs, 2008). Finalmente, las muestras fueron sonicadas durante 1 hora en un baño de ultrasonidos (Cod. 3000513, J.P. Selecta, S.A, Barcelona, España) a temperatura ambiente.

3.2.2.5 Filtración

Después de sonicar, las muestras fueron filtradas con un papel de filtro (número de referencia 1242, 11 cm, Albet) para separar el residuo sólido, que fue lavado 2 veces con 5mL de n-hexano. La disolución fue recogida en un matraz y el volumen fue llevado a 10 mL con ayuda de un rotavapor (Heidolph Laborota 4000 efficient, Alemania) y la adición de la cantidad necesaria de n-hexano.

Materiales y métodos

Fig ura 3.14. Filtrado .

3.2.2.6 Extracción en fase sólida (SPE)

El extracto obtenido después de filtrar contiene una cantidad de grasa muy elevada (Purcaro et al., 2009). Para separar los compuestos de interés (benzo(a)pireno y benzo(a)pireno- d12 (BaP-d12)) se utilizó un procedimiento de extracción en fase sólida (SPE) en un equipo de vacío Supelco Visiprep TM SPE con 12 puertos (Sigma-Aldrich, USA), previamente utilizado para la determinación de HAP en aceites vegetales por Moret y Conte (2002), y comprobada su validez en productos cárnicos ahumados por Purcaro et al. (2009).

Figura 3.15. Equipo SPE.

En primer lugar se lava el cartucho SPE de 5g de sílice (Mega Bond Elut, 20 mL, Varian) con 20 mL de diclorometano durante 2 horas y se seca por completo al vacío. Posteriormente se acondiciona el cartucho con 20 mL de hexano durante 1 hora, evitando en todo momento que se seque. A continuación se carga lentamente (durante 30 minutos aproximadamente) 1mL de la muestra sonicada, diluida con 3 mL de hexano, evitando en todo momento que el cartucho llegue a sequedad.

Capítulo 3

Posteriormente, se eluye la fracción correspondiente a los hidrocarburos alifáticos mediante la adición en el cartucho de 8 mL de una mezcla de hexano y diclorometano 70:30 (v/v). Finalmente se eluye lentamente y hasta sequedad (durante 1 hora) la fracción de HAP, nuevamente con 8 mL de una mezcla de hexano y diclorometano 70:30 (v/v). Ambas fracciones se conservan en tubos especiales para SPE de color ámbar de 15 mL. Se expone a continuación un esquema del proceso.

PROCESO DE EXTRACCIÓN DE HAP MEDIANTE SPE

Figura 3.16. Proceso de extracción en fase sólida, de BaP en productos cárnicos ahumados.

Materiales y métodos

3.2.2.7 Concentración

Se reduce el volumen de muestra (contenida en el vial ámbar de 15 mL procedente de SPE) con una corriente de nitrógeno (situado dentro de una campana de extracción de gases) hasta un volumen un poco inferior a 1 mL, durante aproximadamente 40 minutos. Finalmente se traspasa la muestra al vial de análisis de color ámbar y se ajusta el volumen hasta 1mL. Para comprobar que existe exactamente 1 mL de la mezcla de disolventes en el vial final de análisis, debe pesarse este vacío (antes de llenarlo) y finalmente lleno. Por diferencia de pesadas se comprueba el volumen en el vial. Se encontró que al finalizar el secado, solo permanece hexano en el vial. Por esta razón, para realizar la comprobación mediante pesada, se debe considerar la densidad del hexano (•hexano a 20 ºC: 0,660 g/mL), en lugar de la densidad de la mezcla de hexano y diclorometano 70:30 (v/v) (• mezcla=0,859 g/mL), la cual se calcula mediante la siguiente ecuación:

Ù Ù 1 x Hexane x Dichlorome tan e = + ec.(1) r M r Hexane rDichlorome thane

donde:

rM = densidad de la mezcla.

r hexano = densidad del hexano a 20 ºC: 0,660 g/mL.

rdiclorome tan o = densidad del diclorometano a 20 ºC: 1,325 g/mL.

Ù x hexano = fracción en peso del hexano.

Ù x diclorome tan o = fracción en peso del diclorometano.

Figura 3.17. Vial.

Capítulo 3

3.2.3. PRETRATAMIENTO DE MUESTRAS DE ACEITE

En el pretratamiento de las muestras de aceite se utilizó un procedimiento de extracción en fase sólida (SPE) para extraer el benzo(a)pireno and benzo(a)pireno-d12 (BaP-d12), de acuerdo al descrito por Moret y Conte (2002), similar al previamente descrito para las muestras de chorizo. Se pesaron 2.5 g ± 0.001 g de aceite en un matraz volumétrico de 10 mL y se añadieron 200 µL de disolución de patrón interno benzo(a)pireno-d12 con una concentración de 100 µg/L y se añadió n-hexano hasta llevar el volumen hasta 10mL. Esta disolución fue guardada en refrigeración durante 3 días para permitir que el patrón interno se adaptase a la matriz de la muestra. Entonces, las muestras fueron sometidas a sonicación, durante 1 hora a temperatura ambiente, y 1 mL de la muestra fue sometido al procedimiento SPE, del mismo modo que el descrito en las muestras de producto cárnico.

3.2.4 PRETRATAMIENTO DE LAS TRIPAS

Las tripas fueron pesadas en matraces, se añadieron 20 mL de n-hexano y 200 µL de disolución de patrón interno benzo(a)pireno-d12 con una concentración de 100 µg/L. Esta disolución fue guardada en refrigeración durante 3 días para permitir que el patrón interno se adaptase a la matriz de la muestra. Las muestras fueron tratadas con los mismos pasos que los descritos para el chorizo desde la etapa de sonicación.

3.2.5 DETERMINACIÓN DE BAP MEDIANTE CROMATOGRAFÍA DE GASES/ ESPECTROMETRÍA DE MASAS (GS/MS)

3.2.5.1 Calibración GC/MS

La cuantificación de BaP fue realizada mediante la calibración del equipo GC/MS con disoluciones patrón de benzo(a)pireno y benzo(a)pireno-d12. Las curvas de calibrado fueron generadas inyectando por triplicado 6 disoluciones conteniendo los patrones BaP y BaP-d12, en ciclohexano. Las concentraciones de patrón BaP utilizado en las calibraciones están en el rango desde 0,05 hasta 10 ppb, en función de cada estudio. La concentración de BaP-d12 para todos los viales de calibrado fue siempre 2 ppb. El programa de MS (Agilent Technologies) compara la relación entre la señal analítica de ambos patrones (BaP y BaP-d12) para determinar la

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Materiales y métodos

concentración final. La identificación de los compuestos fue realizada comparando el tiempo de retención de los compuestos en las muestras con los patrones y confirmado verificando que la relación de los ratios de los iones (taget ion/ ion cualificador) estaba dentro del criterio ±20% respecto a la misma relación en los patrones. Un vial blanco (conteniendo solo disolvente), fue introducido entre todos los viales.

3.2.5.2 Cromatografía de Gases (GC)

La separación de BaP y BaP-d12 fue realizada con una columna, de referencia HP-5MS 19091S-433 y dimensiones (30m x 250 •m x 0.25 •m) de Agilent Technologies (Santa Clara, CA, EEUU) en un cromatógrafo de gases (GC) de Agilent Technologies, versión 6890. La temperatura y el volumen de inyección fueron 300 ºC y 1 µL (splitless), respectivamente. Como gas portador se utilizó Helio con un flujo constante de 1,5 mL/min. La temperatura de la rampa utilizada fue: isoterma a 55 ºC durante 1 min, aumento a un ratio de 25 ºC/min hasta 320 ºC durante 3 min, tiempo total 14,60 minutos.

3.2.5.3 Espectrometría de Masas (MS)

La identificación de BaP y BaP-d12 fue realizada mediante el detector espectrómetro de masas (MS) Agilent Technologies 5975 equipado con unas lentes de apertura ultra larga de 6 mm, de referencia G2589-20045. El retraso del disolvente fue de 4 minutos y el voltaje EM (utilizado a voltaje autotune) fue de 1294 voltios. Se utilizó el método SIM con 3 iones específicos (252, 264 and 126), 50 msec dwell/ion. La temperatura del cuadrupolo, de la fuente y de la línea de transferencia fueron 180, 300 y 280°C, respectivamente. Los datos fueron adquiridos utilizando el MS en el modo de monitorización selectiva de los iones (SIM) durante la cuantificación de las muestras y en SCAN (barrido completo) en una primera instancia para identificar y determinar el tiempo de retención de los patrones. Los espectros de los picos fueron comparados con los espectros de masas de los patrones de BaP y BaP-d12 y también con la librería proporcionada por el instrumento. Cada vial fue analizado por triplicado. La determinación de la recuperación obtenida en las medidas se calculó comparando la diferencia entre muestras a las que se añadió una cantidad conocida de patrón BaP (spiked), 1 ppb, y muestras a las que no se añadió patrón (unspiked). Los límites de detección y cuantificación (LOD and LOQ, respectivamente) se

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Capítulo 3

definieron como la concentración de analito que produce una relación señal/ruido (S/N) de 3 y 10 respectivamente. Estos valores fueron también estimados mediante el análisis de muestras blanco y 6 diluciones de patrones de BaP y BaP-d12 en ciclohexano, por triplicado. Se obtuvo la pendiente (m) y la desviación estándar en el origen (sb0) de la respuesta analítica (y) frente a la concentración añadida (x) y LOD y LOQ fueron también estimados mediante las siguientes ecuaciones: LOD=3.3*sb0/m, LOQ=10*sb0/m.

La siguiente tabla resume las condiciones generales de GC/MS

Tabla 3.1 Condiciones generales de cromatografía de gases/espectrometría de masas (GC/MS). Columna: Agilent Technologies HP-5MS 19091S-433 Longitud 30.0 m Diámetro 250 •m Espesor 0, 25 •m Modo Flujo constante = 1,5 mL/min Espectrómetro de masas MSD: Agilent Technologies 5975 BaP Iones: 252 (target),126, 264 (qualifiers) Modo SIM Tiempo de retención: 12.22 min BaP d12 Iones: 264 (target),126, 252 (qualifiers) Tiempo de retención: 12.22 min

Horno : Rampa Min °C/min Siguiente °C Duración Inicial 0 55 1,00 Rampa 1 25 320 3,00 Tiempo total 14,60 min Cromatógrafo de gases : Agilent Technologies 6890 Entrada Splitless, 1 ml Flujo total 34.6 mL/min Temperatura 300 °C Presión 13.00 psi

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Figura 3.18 . Cromatógrafo de gases (GC) 6890/ espectrómetro de masas (MS) 5975, Agilent Technologies.

3.3 CARACTERIZACIÓN FÍSICA DEL EFECTO DE LAS TRIPAS EN EL CHORIZO

3.3.1 CARACTERIZACIÓN DE LAS TRIPAS

3.3.1.1 Determinación de la porosidad

3.3.1.1.1 Pretratamiento

Las tripas sintéticas son proporcionadas por el proveedor en fase seca, mientras que las naturales presentan un elevado contenido de humedad. Mediante porosimetría no es posible el análisis de muestras húmedas. El método de liofilización fue descartado para evitar la posible degradación y/o modificación de las características físicas de las muestras. Por esta razón, se diseñó y preparó un sistema especial para el secado lento y cuidadoso de las tripas, del mismo modo que ocurre en los productos cárnicos, e impidiendo la contaminación de las tripas con los ingredientes.

Para ello se tomaron 60 cm de cada tipo de tripa, natural (de intestino de cerdo) y sintética (de colágeno) y se remojaron en agua con sal. Mientras tanto, se recortó la parte central

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Capítulo 3

de un tubo hueco de cartón de 53 cm, para crear un espacio interior sin material. La superficie del la parte del tubo que permanecía de cartón se recubrió con parafilm para permitir que las tripas deslizasen bien. Entonces se embutieron los palos con las tripas y los sistemas creados se colgaron en barras independientes, las cuales fueron emplazadas en una estantería de metal colocada en una cámara de atmósfera controlada, donde fueron secadas lentamente (del mismo modo que los chorizos) durante 3 días, con unas condiciones de 12ºC de temperatura y 65% de humedad. La figura 3.19 esquematiza el sistema de secado. Una vez secas, las muestras fueron transformadas cuidadosamente, colgadas en barras colocadas en una caja (diseñada también específicamente para el estudio), previniendo el contacto de las mismas con cualquier objeto (ver figura 3.19 b).

(a) (b) Figura 3.19. Procedimiento de secado (a) y transporte (b) de membranas (tripas).

3.3.1.1.2 Determinación de la porosidad

Una vez en estado seco, se tomaron 0,51 g y 0,42 g de las tripas sintética y natural, respectivamente, y se determinó su porosidad mediante porosimetría de intrusión de mercurio utilizando el dispositivo Micromeritics Autopore IV (Norcross, GA, EEUU), de la Unidad de Tecnología Alimentaria de los Servicios Científico Técnicos (SCTs) de la Universidad de Oviedo. Las muestras previamente evacuadas, fueron sometidas a valores de presión en el rango (0,10 - 60.000,00) psia, para obtener las siguientes características de los materiales: datos de intrusión, estructura de los poros y valores de compresibilidad.

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Materiales y métodos

Figura 3.20. Porosímetro Micromeritics Autopore IV.

3.3.1.2 Microscopía electrónica de barrido (SEM)

Se utilizó el microscopio electrónico de barrido (scanning electron microscope, SEM) MEB JEOL-6610LV (Tokyo, Japón) con microanálisis en la Unidad de Microscopia Electrónica de los (SCTs) de la Universidad de Oviedo, para estudiar ambas caras de las tripas natural y sintética, pretratadas como describe el apartado 3.3.1.1.

Figura 3.21. Microscopio electrónico de barrido MEB JEOL-6610LV SCTs UO.

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Capítulo 3

3.3.1.3 Estereomicroscopía de fluorescencia óptica

Se utilizó el Microscopio de Fluorescencia Óptica Leica M205 FA (Wetzlar, Alemania) ubicado en la Unidad de Microscopía Fotónica y Proceso de Imágenes de los SCTs de la Universidad de Oviedo, para estudiar la morfología de las siguientes muestras: ambas caras de las tripas natural y sintética, pretratadas como describe el apartado 3.3.1.1 y sin pretratar

Figura 3.22. Microscopio de Fluorescencia Óptica Leica M205 FA. SCTs UO.

3.3.2 CHORIZOS EMBUTIDOS EN TRIPAS

3.3.2.1 Determinación del color

Se determinó el color de la superficie exterior de 36 chorizos (18 embutidos en cada tipo de tripa, natural y sintética), a los 0, 1, 4, 5, 7 y 11 días de ahumado fabricados como especifica el apartado 3.1.1.1. Cada día se tomaron 3 muestras de chorizo embutido en cada tripa. El color de las diferentes muestras fue determinado en el espacio de color CIELAB (L*, a*, b*) mediante el espectrofotometro UltraScan VIS (HunterLab) de la Unidad de Tecnología Alimentaria de los Servicios Científico Técnicos de la Universidad de Oviedo. El equipo fue calibrado utilizando como patrones una teja blanca y una trampa de luz y se comprobó su correcta calibración utilizando como referencia una teja verde. En la medición se empleo una cubeta de cuarzo de 10 mm de paso de luz (USVIS1034). Los análisis fueron realizados en reflectancia con el método de reflexión especular excluida, este método elimina los errores generados por la turbidez y la bruma de la

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Materiales y métodos

muestra, de manera que la evaluación del color es mas similar a la percepción del ojo humano (HunterLab, 2015). Los valores L*, a* y b* corresponden a claridad, (-L) negro o (+L) blanco, verde (-a) o rojo (+a) y azul (-b) o amarillo (+b), respectivamente. Las medidas de color se realizaron a temperatura ambiente (20 ± 2 ºC). Los parámetros de color se midieron 30 veces para cada chorizo estudiado. El cambio total de color (•E; Eq. (1)) , ángulo de tonalidad cromática (Eq. (2)), chroma (índice de saturación; Eq. (3)) e índice de pardeamiento ó enmarronecimiento (browning index) (BI; Eq. (4)) se calcularon utilizando los valores Hunter L, a, y b (Homco-Ryan et al., 2004; Laca, Sáenz, Paredes, & Díaz, 2010; Maskan, 2001), de la siguiente manera:

Eq.(2)

Hue angle= Ec. (3)

Chroma= Ec. (4)

Ec. (5) donde:

Ec. (6)

Fig 3.23. Colorímetro.

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Capítulo 3

3.3.2.2 Determinación de la textura

Se determinó la textura de la superficie exterior de 36 chorizos (18 embutidos en cada tipo de tripa, natural y sintética), a los 0, 1, 4, 5, 7 y 11 días de ahumado fabricados como especifica el apartado 3.1.1.1. Cada día se tomaron 3 muestras de chorizo embutido en cada tripa. Para ello se utilizó el analizador de Textura TA.XTPlus. Los chorizos se colocan enteros (sin trocear) en una plataforma metálica. Se utilizó una sonda esférica de 1/2 pulgada de diámetro, de acero inoxidable para mediar la fuerza de penetración de la sonda en el producto cárnico. En los ensayos se determinaron tanto la textura como la pegajosidad.

Figura 3.24. Texturómetro.

3.3.2.3 Determinación del contenido de humedad

El extracto seco total de las muestras se determinó por gravimetría, por triplicado, durante 0, 1, 4, 5, 7 y 11 días de ahumado de ChN y ChS. Para ello, se pesaron aproximadamente 20 gramos de arena de mar gruesa, de referencia 211161 (Panreac, Spain), en morteros de acero inoxidable con su varilla, y se mantuvieron en un desecador durante 30 minutos, hasta alcanzar un valor de masa constante. A continuación, se pesaron aproximadamente 6 gramos de muestra en el mortero, y se homogeneizaron con la arena con la ayuda de la varilla.

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Materiales y métodos

Figura 3.25. Pocillos.

El sistema (constituido por el mortero, la arena, la muestra y la varilla) se introdujo en la estufa (Memmert, Germany) a 105±2ºC durante 5 horas. Entonces, el sistema se dejó enfriar en un desecador de vacio a temperatura ambiente, durante 30 minutos aproximadamente, y finalmente se pesó.

Figura 3.26. Dispositivo para determinación de humedad: desecador, arena, pocillos y horno.

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Capítulo 3

3.3.2.4 Determinación de la estructura

3.3.2.4.1 Microscopía electrónica de barrido (SEM)

Se utilizó el microscopio electrónico de barrido (scanning electron microscope, SEM) MEB JEOL-6610LV con microanálisis (ver figura en apartado 3.3.1.2) de la Unidad de Microscopia Electrónica de los Servicios Científico Técnicos (SCTs) de la Universidad de Oviedo (UO), para estudiar las tripas natural y sintética de 8 chorizos después de 3 y 5 días de ahumado, fabricados como describe el apartado 3.1.1.1 y pretratadas mediante congelación a -80ºC durante 4 horas y secadas en un liofilizador (Telstar-Cryodos, España) durante un día.

3.3.2.4.2 Estereomicroscopía de fluorescencia óptica

Se utilizó el Microscopio de Fluorescencia Óptica Leica M205 FA (ver figura en apartado 3.3.1.3) de la Unidad de Microscopía Fotónica y Proceso de Imágenes de los Servicios Científico Técnicos de la Universidad de Oviedo, para estudiar la morfología de las tripas (superficie exterior) natural y sintética de 8 chorizos sin ahumar (4 embutidos en cada tipo de tripa) y de 40 chorizos (fabricados como especifica el apartado 3.1.1.1), ahumados entre 0 y 10 días (20 embutidos en cada tipo de tripa) y sus diferentes profundidades, externa (una vez que la tripa fue retirada), e interna.

3.4 ANÁLISIS ESTADÍSTICOS

Los datos obtenidos en al presente tesis doctoral, fueron tratados con el paquete estadístico Statgraphics Plus 3.1, para Windows 3.0® (Statpoint Technologies, Inc. Warrenton, Virginia, EEUU), tal y como se detalla en cada trabajo. Se realizaron pruebas “ t-S tudent” para comparar entre las medias de las muestras, y determinar si presentaban diferencias estadísticamente significativas, bajo un criterio de p-valor<0.05. Para comparar conjuntos de datos obtenidos entre diferentes muestras, se aplicaron análisis “multirango” . Previamente se estudió si la distribución estadística correspondía a una distribución normal, estudiando las medidas de asimetría, el coeficiente de asimetría de Fisher, junto con las medidas de apuntamiento o curtosis. Se realizó la prueba F de Fisher (F-test), para comparar las desviaciones estándar de las muestras.

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Resultados• • • • • • • • • Resultados

•4.•RESULTADOS•

4.14 .1 DETERMINACIÓN DE B(a)P EN CHORIZOS AHUMADOS DEL PRINCIPADO DE ASTURIAS

Como se ha indicado en la presente memoria (apartado 1.1) ante las perspectivas de crecimiento de consumo mundial de carne y productos cárnicos ahumados, diversas entidades (Organización de las Naciones Unidas para la Alimentación y la Agricultura (FAO), Organización Mundial de la Salud (OMS), etc.) alertan sobre la necesidad de evaluar y controlar la presencia de HAP cancerígenos, representados por el BaP, en productos cárnicos ahumados de todo el mundo. Por todo ello, recientemente la legislación europea ha reducido la cantidad máxima permitida de HAP y BaP en estos alimentos.

La elevada tasa de atomización del sector cárnico español obliga a las pequeñas (y micro) empresas cárnicas a seguir utilizando el método de ahumado directo, como método para secar sus productos, prolongando su vida útil, y conferir buenas propiedades organolépticas. Sin embargo, las referencias científicas han demostrado que este método es susceptible de conferir estas sustancias cancerígenas a los productos.

En el siguiente artículo de investigación se presentan los resultados sobre la puesta a punto de un método analítico para la determinación de BaP en chorizo asturiano ahumado. Así mismo, se evalúa el contenido de BaP de chorizos asturianos, elaborados por 16 empresas diferentes del Principado de Asturias, recogidos en establecimientos de venta al público. Se llevó por tanto a cabo un estudio que nunca había sido realizado en esta provincia española por la comunidad científica. En el este artículo se estudió la relación entre el contenido de BaP y la humedad de los chorizos asturianos, evaluando la calidad del ahumado directo como método de secado, así como la adecuación de los chorizos ahumados del Principado de Asturias a la nueva normativa europea.

Publicación: Ledesma, E., Rendueles, M., & Díaz, M. (2015). Spanish smoked meat products: Benzo(a)pyrene (BaP) contamination and moisture. Journal of Food Composition and Analysis, 37, 87 –94.

Situación: Artículo publicado en Journal of Food Composition and Analysis (Elsevier).

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Journal of Food Composition and Analysis 37 (2015) 87–94

Contents lists available at ScienceDirect

Journal of Food Composition and Analysis

jo urn al ho mepag e: ww w.els evier .c om /lo cat e/jfc a

Original research article

Spanish smoked meat products: Benzo(a)pyrene (BaP) contamination

and moisture

a,b a a,

Estefanı´a Ledesma , Manuel Rendueles , Mario Dı´az *

Department of Chemical Engineering and Environmental Technology, University of Oviedo, Faculty of Chemistry, C/Julia´n Claverı´a s/n, 33071 Oviedo,

Principado de Asturias, Spain

Research, Development and Innovation (R&D+i) Department, El Ho ´rreo Healthy Food S.L., C/Las Caban˜as 43-45, 33180 Noren˜a, Principado de Asturias, Spain

A R T I C L E I N F O A B S T R A C T

Article history: Traditional direct smoking is used for drying and ﬂavouring foodstuffs, although carcinogenic

Received 21 October 2013

compounds are added during this process, namely polycyclic aromatic hydrocarbons (PAH). The

Received in revised form 8 September 2014

maximum permissible content of benzo(a)pyrene (BaP) (a current marker for the occurrence and effect

Accepted 30 September 2014

of PAH in foods) in smoked meat products was reduced from 5 to 2 mg/kg on 1/09/2014, in compliance

Available online 28 October 2014

with European Regulation No. 835/2011. In this study, an analytical method has been developed to

determine BaP content consisting of PAH extraction assisted by sonication followed by solid-phase

Keywords:

extraction sample clean-up and analytical determination using gas chromatography/mass spectrometry.

Food composition

Sixteen commercial chorizo samples from 16 different Spanish producers from the Principality of

Food analysis

Asturias were studied. Five of the samples exceeded the 2 ppb BaP limit. The relationship between

Benzo(a)pyrene (BaP)

moisture and BaP content in chorizo was examined, in order to verify the quality of the manufacturing

Polycyclic aromatic hydrocarbons

PAH process. Moisture content did not correlate with BaP content in chorizo.

Smoked meat products

Gas chromatography/mass spectrometry GC/MS

Solid-phase extraction SPE

Food safety

1. Introduction Humans are exposed to PAH in three ways: through food, the

environment and a combination of both, i.e. food contaminated

Food smoking is an old and traditional technological process due to exposure to the environment. The main source of PAH is diet

widely applied to many foodstuffs such as meat, ﬁsh and cheese, (Lodovici et al., 1995; Phillips, 1999; Falco´ et al., 2003; Iba´n˜ez et al.,

not only for the special organoleptic proﬁles that it confers, but also 2005), contributing to more than 70% of total exposure in non-

due to the inactivating effect of smoke and heat on enzymes and smokers (Gilbert, 1994; McGrath et al., 2007). PAH enter the food

microorganisms (Simko, 2002; Codex Alimentarius CAC/RCP 68/ chain by means of a large variety of unprocessed and processed

2009). However, unwanted compounds are produced during foods, due to their processing, packaging and thermal processes

traditional uncontrolled smoking processes because of incomplete such as smoking, drying, roasting, baking, barbecuing and frying

combustion or thermal decomposition of the organic material (Codex Alimentarius CAC/RCP 68/2009). The major contributors to

employed (wood) (Djinovic et al., 2008; Danyi et al., 2009). These PAH intake are cereals and cereal products (owing to high

compounds are polycyclic aromatic hydrocarbons (PAH), whose consumption in diets) and vegetable fats and oils (due to higher

carcinogenic, mutagenic and bioaccumulative capacities have been concentrations of PAH in this food group) (Codex Alimentarius

reported by the World Health Organisation (WHO), the Interna- CAC/RCP 68/2009).

tional Agency for Research on Cancer (IARC), the European Food Particular attention has been paid to smoked meat products,

Safety Authority (EFSA) and the US Environmental Protection because the highest levels of total PAH have been detected in these

Agency (EPA). foods (Larsson et al., 1988; Gomaa et al., 1993; Karl and Leinemann,

1996; Martorell et al., 2010). The Commission of the European

Communities (EC) has recently amended Regulation (EC) No. 1881/

2006 by means of Regulation (EU) No. 835/2011 of 19 August 2011,

* Corresponding author. Tel.: +34 985103439; fax: +34 985 103434.

E-mail address: [email protected] (M. Dı´az). setting new maximum levels for PAH in foodstuffs. New maximum

http://dx.doi.org/10.1016/j.jfca.2014.09.004

88 E. Ledesma et al. / Journal of Food Composition and Analysis 37 (2015) 87–94

levels for the sum of four substances (PAH4) (benzo(a)pyrene, sodium nitrite and other food additives are used to ensure the

benzo(a)anthracene, benzo(b)ﬂuoranthene and chrysene) were microbiological stability and quality of the foodstuff.

introduced, maintaining a separate maximum level for BaP, to In this study, 16 samples of chorizo were analysed. All the

ensure comparability between previous and future data. The samples were bought from a market. Each sample came from a

maximum permissible BaP content in smoked meat and smoked different manufacturer in Asturias (a region in the north of Spain).

meat products was 5.0 mg/kg in wet weight until 31/8/2014 and The samples were purchased and analysed between 15th March

has been 2.0 mg/kg in wet weight since 1/9/2014. The permissible and 25th November 2012.

sum of PAH4 in these foods was 30.0 mg/kg in wet weight from 1/9/ Samples A, C, F, I, J, M, O and P are labelled as ‘‘extra’’ quality and

2012 to 31/8/2014 and 12.0 mg/kg in wet weight since 1/9/2014. samples G, H, K and N as ‘‘top’’ quality products. According to

Likewise, the ‘‘ Code of practice for the reduction of contamina- Spanish regulations, the maximum amount of fat, hydroxyproline

tion of food with (PAH) from smoking and direct drying processes’’ and total carbohydrates (expressed in terms of glucose content) in

(CAC/RCP 68/2009) stipulates that PAH contamination in foodstuffs ‘‘extra’’ quality chorizos is 57%, 0.8% and 8%, respectively, while the

via food processing should be controlled, taking into consideration minimum amount of meat proteins allowed in these samples is

the food processing technology. The food producer should be aware 30%, expressed in terms of dry matter (DM) content. The maximum

of the conditions under which higher levels of PAH are generated amount of fat, hydroxyproline and total carbohydrates (expressed

and, wherever possible, should control those conditions to minimise in terms of glucose content) in ‘‘top’’ quality chorizos is 60%, 0.7%

their formation. To accomplish this, an analysis of important points and 9%, respectively, and the minimum amount of meat proteins

to consider in processes used or intended to be used in food allowed in these samples is 26%, expressed in DM content. The

production with smoking or direct drying should be carried out. maximum permissible moisture content for all samples is 45%. The

Both bodies, EU regulators and the Codex Alimentarius, conclude standard amount of fat, meat proteins and carbohydrates, in

that more scientiﬁc data are needed to elucidate the precise ‘‘extra’’ and ‘‘top’’ quality Spanish chorizos is 57–60%, 26–30% and

inﬂuence of the smoking method employed and other processing 8–9%, respectively. Once collected, the samples were stored in the

parameters on PAH contamination of food. A variety of both old and dark at –18 C.

modern pre-treatment and analytical methods has been used to

determine PAH in foods. Old pre-treatment methods, like saponiﬁ- 2.2. Reagents and standards

cation, liquid–liquid extraction and Soxhlet in combination with gel

permeation chromatography (GPC), are time consuming and involve Benzo[a]pyrene (BaP) analytical standard solution was supplied

high solvent consumption. Modern methods, such as microwave- by Sigma–Aldrich (Kempton Park, South Africa) at a concentration

assisted extraction (MAE), accelerated solvent extraction (ASE) of 100 mg/mL in cyclohexane and a total volume of 2 mL.

solid-phase microextraction (SPME) and direct extraction devices Benzo[a]pyrene internal standard (d 12 , 98%) (BaP d-12) solution

(DED) require the use of expensive technologies. The combination of was obtained from Cambridge Isotope Laboratories, (Andover, MA)

sonication with solid-phase extraction has been tested in some at a concentration of 200 mg/mL in isooctane and a total volume of

foodstuffs, affording results that are both fast and inexpensive. 1.2 mL.

Typical analytical methods for PAH identiﬁcation are: high pressure All solvents for sample preparation, hexane, dichloromethane,

liquid chromatography (HPLC) combined with ﬂuorescence (FLD) or cyclohexane and isooctane (2,2,4-trimethylpentane), were pur-

ultraviolet (UVD) detectors and gas chromatography (GC) combined chased from Sigma–Aldrich with ACS reagent purities 99% (GC)

with ﬂame ionisation (FID) or mass spectrometry (MS). GC/MS is grade.

widely used nowadays to determine PAH in foodstuffs (Moret and Solid phase extraction (SPE) cartridges were supplied by Varian

Conte, 2002; Simko, 2002; Martin and Ruiz, 2007; Purcaro et al., Iberica (Madrid, Spain) and Agilent Technologies (Santa Clara, CA).

2009 ). The Mega Bond Elut cartridges employed contain a silica sorbent

The main aims of this study were to determine the BaP content phase, with a particle size of 40 nm, a weight of 5 g and a volume of

of a traditional Spanish meat product called chorizo in an as-yet 20 mL.

unstudied region, the Principality of Asturias, and to test whether Amber-coloured ﬂasks and vials were used to protect the PAH

this content will comply with the recently modiﬁed maximum BaP from light decomposition. Speciﬁcally, 15 mL (21 mm Â 70 mm)

content permitted by EU regulation No. 835/2011. An analytical amber glass vials were purchased from Sigma–Aldrich for PAH

method based on a combination of sonication, SPE and GC/MS was collection during the SPE step.

developed and employed for this purpose. Furthermore, the

manufacturing method used for each analysed product was 2.3. Apparatus

studied to evaluate the quality of drying versus BaP content in

the ﬁnal meat product. Finally, the traditional meat product An ultrasonic bath (J.P. Selecta, S.A., Barcelona, Spain) and a

manufacturing method was evaluated to test whether any changes Supelco Visiprep SPE vacuum manifold with 12 ports (Supelco,

are required to achieve high quality, safe and healthy foods. Bellefonte, PA) were used for each meat product sample pre-

treatment.

An Agilent Technologies 5975 mass spectrometer (MS) coupled

2. Materials and methods to an Agilent Technologies 6890 gas chromatograph (GC) was used

for BaP analytical identiﬁcation. An Agilent Technologies HP-5MS

2.1. Samples (30 m Â 0.25 mm Â 0.25 mm) GC column was chosen for this

study.

Chorizo is a typical Spanish smoked meat product made with the

following ingredients: pork meat, pork fat, salt, garlic, sweet or spicy 2.4. Sample pre-treatment

paprika and herbs minced, mixed and sheathed in a natural casing

(or skin) made from animal intestine. Once prepared, chorizo is 2.4.1. Grinding step

smoked for 7–15 days, usually employing a mixture of oak and First, the skin of the chorizo was removed according to the

chestnut woods for this purpose. Nowadays, antioxidants such as procedures speciﬁed in Fretheim (1976) and Wretling et al.

sodium citrate and sodium ascorbate, colourings such as carmine, (2010). Then, 200 g of chorizo were ﬁnely homogenised in a meat

emulsiﬁers such as sodium triphosphate, food preservatives such as grinder (Minirobot D81 meat grinder; Moulinex, France) following

E. Ledesma et al. / Journal of Food Composition and Analysis 37 (2015) 87–94 89

the protocol devised by Purcaro et al. (2009). Pre-treatment of all (rMixture = 0.859 g/mL), that is calculated with the theoretical

samples was carried out in triplicate. following equation:

2.4.2. Lyophilisation step 1 xˆ : xˆ

¼ Hexane þ Dichlorometane :

Three aliquots of 20 g chorizo were placed on individual

rM rHexane rDichloromethane

lyophilisation plates, carefully sealed with aluminium foil, and

weighed before freeze-drying. Each sample was then frozen to – where rM is the density of the mixture; rHexane is the density of

8 8

80 C for 4 hours and freeze-dried (Telstar-Cryodos, Spain) for hexane at 20 C: 0.660 g/mL; rdichlorometane is the density of

8 1 day. Finally, the plates (containing the sample) were weighed dichloromethane at 20 C: 1.32 g/mL; xˆHexane is the weight fraction

after freeze-drying. of hexane; Dichloromethane is the weight fraction of dichloromethane.

2.4.3. Sonication step 2.5. Analysis

Two grams of freeze-dried chorizo (one from each lyophilisa-

tion plate) were placed in ﬂasks (the remaining lyophilised chorizo 2.5.1. GC/MS calibration

was stored in a freezer). Subsequently, 20 mL of n-hexane, 200 mL BaP quantiﬁcation was performed via prior calibration of the

of benzo[a]pyrene-d12 internal standard solution at a concentra- GC/MS system with benzo(a)pyrene and benzo[a]pyrene-d12

tion of 100 mg/L and 100 mL of benzo[a]pyrene standard solution standard solutions. For calibration purposes, benzo[a]pyrene-d12

at a concentration of 100 mg/L were added (dilutions from the standard solution was added to check the extraction recovery

stock solution were made previously). This solution was carefully during pre-treatment methods.

stored under refrigeration for 3 days to help the deuterated Calibration curves were generated with the Chemstation

compound adapt to the sample matrix. This is the appropriate software of the GC/MS by injecting 6 diluted standard solutions

point to add the internal standard compound as it is the ﬁrst time of BaP and BaP-d12 in cyclohexane, in triplicate. Calibration curves

both compounds are in the same matrix (Stumpe et al., 2008; were deﬁned as amount ratios (x) (concentration of BaP standard

Purcaro et al., 2009). Finally, the samples were sonicated for 1 hour divided by the concentration of the BaP-d12 standard) versus

at ambient temperature. response ratios (y), (ratios of BaP response divided by BaP-d12

standard response). BaP standard concentrations for each calibra-

2.4.4. Filtration tion vial were 0.05 ng/mL, 0.10 ng/mL, 0.50 ng/mL, 1.00 ng/mL,

After sonication, the samples were ﬁltered through ﬁlter paper 2.00 ng/mL and 5.00 ng/mL. The concentration of BaP-d12 for all

to remove the solid meat waste, which was washed with additional calibration vials was 2.00 ng/mL. BaP-d12 and BaP concentrations of

n-hexane (2 Â 5 mL). The solvent was subsequently collected in a the calibration method were ﬁtted to the range of BaP concentra-

ﬂask and the volume was made up to 10 mL with the help of a tions found in the samples. The MS Assistant programme (Agilent

rotatory evaporator (Heidolph Laborota 4000; Schwabach, Technologies) compares the relationship between both BaP

Germany) and the addition of the necessary amount of n-hexane. standard signals (BaP and BaP-d12) to determine the ﬁnal BaP

concentration. A blank (i.e. a vial containing only the solvent

2.4.5. Solid-phase extraction (SPE) cyclohexane, with no BaP and BaP-d12 standard compounds) was

The extract obtained after sonication contained an appreciable also included among all the standard vials containing BaP

amount of fat (Purcaro et al., 2009). A solid-phase extraction (SPE) compounds.

procedure was used to isolate benzo(a)pyrene and benzo[a]pyr-

ene-d12 (BaP-d12) (the sought after PAH in the sample), similar to 2.5.2. Gas chromatography (GC)

the previous setup for rapid PAH determination in vegetable oils by Separation of BaP and BaP-d12 was performed on an HP-5MS

Moret and Conte (2002). column (30 m Â 0.25 mm Â 0.25 mm; Agilent Technologies). The

First, a 5-g silica SPE cartridge (Mega Bond Elut, 20 mL, Varian, injection temperature and volume were 300 C and 1 mL (splitless),

Palo Alto, CA) was slowly washed with 20 mL of dichloromethane, respectively. Helium was applied as carrier gas at a constant ﬂow

dried completely under vacuum and conditioned with 20 mL of n- rate of 1.5 mL/min. The temperature ramp used was the following:

8 8

hexane (to prevent the cartridge from drying). Next, 1 mL of the isothermal at 55 C for 1 min, increasing at a rate of 25 C/min to

sonicated sample was diluted in 3 mL of n-hexane and slowly 320 C for 3 min; total run time was 14.60 min.

loaded onto the cartridge, avoiding complete drying of the

cartridge. The aliphatic hydrocarbons were then discharged via 2.5.3. Mass spectrometry (MS)

the addition of 8 mL of a mixture of hexane and dichloromethane Identiﬁcation of BaP and BaP-d12 was performed using an

70:30 (v/v) to the cartridge. Finally, after loading the cartridge with Agilent Technologies 5975 mass spectrometer with a G2589-20045

another 8 mL of the hexane and dichloromethane 70:30 (v/v) 6-mm ultra-large aperture drawout lens. The solvent delay was

solution, the PAH, BaP and BaP-d12 contained in the sample were 4 min and the electron multiplier (EM) voltage was 1294 V. The

8 8

slowly discharged into 15-mL amber glass vials, allowing the quadrupole temperature was 180 C, the source temperature 300 C

cartridge to dry under vacuum. and the transfer line temperature 280 C. Full scan spectrum analysis

(scan) mode method was used to obtain the full mass spectra of

2.4.6. Concentration step benzo(a)pyrene and benzo[a]pyrene-d 12 standards, using a concen-

The samples were concentrated after the SPE step, the volume trated solution of 10 mg/mL. Scan mode was used to identify, and

being reduced to less than 1 mL using a nitrogen stream at the top select target and main qualiﬁer ions and the retention time (RT):

of the 15-mL vials. Finally, the samples were loaded into 2 mL GC/ BaP: RT 12.22 min, target ion: 252, qualiﬁer ions: 264, 126; BaP-d12:

MS amber glass vials and the ﬁnal volume was up to 1 mL with the RT 12.20 min, target ion: 264, qualiﬁer ions: 252, 126. These data

addition of the necessary volume of solvent calculated by weighing were used to create the selected ion monitoring (SIM) method

the vials and the application of the density of the solution. It was applied for the analysis of all calibration vials and samples. 50 ms

found that during the ﬁnal concentration step only hexane remains dwell/ion was applied in SIM mode. Peaks spectra were compared to

in the sample. For this reason, in this step the density of hexane the mass spectra of BaP and BaP-d12 standards. Qualiﬁer-to-target

(rHexane = 0.66 g/mL) must be applied instead of the density of the ion ratio criteria were Æ10% compared to the standard’s ratio for

mixture of hexane and dichloromethane 70:30 (v/v) solution compounds conﬁrmation and assessment of spectral interferences.

90 E. Ledesma et al. / Journal of Food Composition and Analysis 37 (2015) 87–94

Matrix-effect assessment criteria were deﬁned as a Æ20% ratio of target and BaP standards were added just before sonication and left to

ions of BaP and BaP-d12 in samples compared to standards. Detection stand for 3 days, allowing all the analytes to adapt to the liquid

and quantiﬁcation limits (LOD s and LOQ s, respectively) were evaluated matrix. Flasks were stored under refrigeration in the dark to

on the basis of noise obtained with the analysis of the blank samples. prevent PAH decomposition due to light exposure (Simko, 2002).

LOD and LOQ were deﬁned as the concentration of the analyte that Figs. 1 and 2 show the ion chromatograms of the spiked and the

produced a signal-to-noise ratio of 3 and 10, respectively. Recoveries unspiked sample, respectively. In the lower line, the peak of the

were calculated by comparing the difference between spiked and target ion 264 of BaP-d12 can be detected at an RT of 12.20 min,

unspiked samples with the known amount of added BaP standard and in the upper line the peak of the target ion of BaP 252 can be

(1 ppb). detected at an RT of 12.22 min. The arrow indicates the BaP peak

(added compound), which increases in the spiked sample in

2.6. Statistical analyses comparison with the unspiked one, and its relationship with the

BaP-d12 standard peak.

Experimental data were evaluated by running analysis of The combination of the sonication and SPE pre-treatment

variance (ANOVA), which determined whether there were any methods to determine the BaP content in smoked meat products

signiﬁcant differences between means at a 95% conﬁdence level. by GC/MS analysis was found to be a good method, as it entails a

Multiple range test was used to distinguish which means were low level of solvent consumption and waste generation, short

signiﬁcantly different. Standardised skewness and standardised operating times and is easy to set up.

kurtosis were used to assess if the samples came from normal

distributions. These analyses were performed using STATGRAPHICS 3.2. BaP content

PLUS for Windows 3.0 (StatPoint, Inc., Herndon, VA, USA).

All the samples were successfully analysed by the proposed

3. Results and discussion method. Fig. 3 shows, as an example, the ion chromatogram of a

directly smoked chorizo (sample A). Fig. 4 shows the moisture and

3.1. Procedure performance BaP content (mg/kg) found in the 16 analysed direct smoked meat

products. The samples were alphabetically identiﬁed according to

The basic analytical method of Purcaro et al. (2009) was applied. their order of analysis. In this ﬁgure, horizontal bold lines have

Some modiﬁcations were implemented to adapt the method for its been used to indicate the BaP content limit speciﬁed by the EU

application to the analysis of chorizo samples and GC/MS regulation before 31/8/2014, i.e. 5 mg/kg (upper line), and the

determination. Table 1 shows benzo(a)pyrene identiﬁcation ions current regulation, applied from 1/9/2014, i.e. 2 mg/kg (lower line).

(target compound and qualiﬁer ions), retention time, limits of

detection (LOD) and quantiﬁcation (LOQ) in ng/mL, instrument 3.2.1. Moisture and BaP content in smoked meat products

linear dynamic ranges, determination coefﬁcient (r ) and recover- One of the most important goals of the smoking process

y Æ repeatability for a spiked sample. Target ions ratios and qualiﬁer- during chorizo manufacturing is drying. The water content of

to-target ion ratios found in all samples were within the deﬁned meat products decreases during this process. This reduction in

criteria (Æ20%). moisture content is important, as it has an inactivating effect on

One of the key features of the procedure performance is the microbial growth, hence extending the shelf life. However, as the

deﬁnition of the appropriate moment to add the standards. All Codex Alimentarius CAC/RCP 68/2009 speciﬁes and some

analytes, both those deliberately added (BaP and BaP-d12 scientiﬁc studies conﬁrm, the PAH content in meat products

standards) and native (original BaP content in the sample), must generally increases during the smoking process (Djinovic et al.,

be ﬁrmly and equally bound to the sample under the same 2008 ). As the Codex Alimentarius advises, the smoking process

conditions. If not, recoveries will be incorrect. An appropriate should be optimised to obtain smoked products with a low PAH

reference material must be used for the method to be accurate content, but a good microbiological status, organoleptic proper-

and to achieve precision testing, i.e. one with a similar sample ties in the ﬁnal product. The ideal method should have no adverse

matrix and whose content in the target analytes has been effects on the product’s appearance, ﬂavour, taste or nutritional

certiﬁed. There is no suitable certiﬁed material for chorizo. In properties.

these cases, a spiking procedure must be used to deﬁne As Fig. 4 and Table 2 show, there is little relationship between

recoveries (Purcaro et al., 2009 ). moisture and BaP content in the analysed smoked meat products.

The pre-treatment method was accordingly applied to a 2.00-g ANOVA indicated that there is a statistically signiﬁcant difference

aliquot of chorizo smoked by means of traditional combustion. between the means of the 16 variables at the 95.0% conﬁdence

During the analytical steps, the best moment to add the BaP-d12 level. In order to determine which means are signiﬁcantly different

standard is just before sonication, as this is when all the analytes from others in the whole analysis of the samples range, multiple

(the natural analyte in the sample and the added BaP) will be in the range test was applied to the results. This test showed

same matrix, i.e. a liquid matrix. For this reason, hexane, BaP-d12 10 homogeneous groups with signiﬁcant differences between

Table 1

Benzo(a)pyrene (BaP) conﬁrmation ions, retention time, limits of detection (LOD) and quantiﬁcation (LOQ) in ng/mL, instrument linear dynamic ranges, determination

coefﬁcient (r ) and recovery Æ repeatability.

Determined Ions Retention LOD LOQ Instrument linearity Recovery assays

compound time (min) (ng/mL) (ng/mL)

Standards r Added Recovery Æ RSD (%) range concentration (ng/mL)

range (ng/mL)

Benzo(a)pyrene Target: 252 12.22 0.05 0.24 0.05–5 0.999 1.00 98 Æ 1.04 to 102 Æ 3.70

Qualiﬁers: 264, 126

(LOQ) in mg/L (n = 8).

E. Ledesma et al. / Journal of Food Composition and Analysis 37 (2015) 87–94 91

Fig. 1. 1.0 ng/mL Benzo(a)pyrene (BaP) spiked sample of chorizo.

each other (p < 0.05). As can be seen in Fig. 4, the sample with the 3.2.2. BaP content in smoked meat products with regard to the

highest BaP content in this study has the lowest moisture content regulation limit

(sample O). This could suggest that when the moisture content As can be seen in Fig. 4, the chorizo samples analysed in this

decreases, the BaP content increases, which could occur when study present a BaP content ranging between less than 0.38 and

increasing smoking time, in accordance with other studies 3.21 mg/kg in wet weight. This means that all the studied samples

(Djinovic et al., 2008). However, the other results obtained in comfortably met the previous legal limit set at 5 mg/kg in wet

the present study indicate that chorizos with a similar BaP content weight. However, 5 of them (31% of the samples) do not meet the

can have very different moisture contents. new limit of 2 mg/kg applicable from 1/9/2014. Fig. 5 summarises

In conclusion, the smoking process can be adapted to obtain the average, maximum and minimum BaP contents (mg/kg) found

smoked meat products with a low moisture content which present in direct smoked samples of the tested products. The results

a good microbiological status and a low BaP content. The obtained in this study indicate that smoked meat product

traditional direct smoking process is affected by a large number manufacturers should make some modiﬁcations in the smoking

of parameters, making it difﬁcult to avoid PAH contamination of process to offer healthy smoked meat products in compliance with

foods (Djinovic et al., 2008; Stumpe et al., 2008; Lorenzo et al., EU regulation No. 835/2011.

2011; Santos et al., 2011; Roseiro et al., 2012). However, producers In itschapter ‘‘Code of practice for the reduction of contamination

should study and control their own traditional processes to make of food with (PAH) from smoking and direct drying processes’’

healthier foods, as explained in the following sections. (CAC/RCP 68/2009), Codex Alimentarius points out that adaptations

Fig. 2. Unspiked sample of chorizo.

92 E. Ledesma et al. / Journal of Food Composition and Analysis 37 (2015) 87–94

Fig. 3. 252 Benzo(a)pyrene (BaP) and 264 Benzo(a)pyrene (BaP D12) ions chromatograms of a 1.0 ng/mL BaP spiked sample of direct smoked chorizo (sample A).

of current smoking technology may be necessary in some cases. It the food and the heat source, the position of the food in relation to

stipulates that PAH contamination in foodstuffs via food processing the heat source, the fat content of the food and what happens during

should be controlled taking into account the chosen food processing processing, the duration of smoking and direct drying, the

technology. In order to regulate PAH formation during smoking and temperature during smoking and direct drying, the cleanliness

direct drying, the code recommends controlling a number of and maintenance of equipment and the design of the smoking

variables. These include the kind of fuel, smoking or drying method chamber and the equipment used for the smoke/air mixture. It

(direct or indirect), the process of generating smoke in relation to the especially recommends replacing direct smoking processes (tradi-

temperature of pyrolysis and to airﬂow in the case of a smoke tional smoking methods by means of wood combustion) by indirect

generator (friction, smouldering, thermostated plates) or in relation ones, such as the use of a friction smoke generator. Only a few of

to other methods, such as direct smoking or regenerated smoke by these factors have been studied. For instance, it has been

atomising smoke condensate (liquid smoke), the distance between demonstrated that the kind of wood used for smoking meat

Fig. 4. Benzo(a)pyrene (BaP) content (mg/kg) and moisture (%) of direct smoked chorizo and comparison with the EU Regulation limit (n = 3).

E. Ledesma et al. / Journal of Food Composition and Analysis 37 (2015) 87–94 93

Fig. 5. Benzo(a)pyrene (BaP) content (mg/kg) in direct smoked chorizos from the north of Spain and comparison with the EC Regulation limit.

products has a significant influence on the amount of PAH in the final 3.2.3. Improving the traditional smoked meat product smoking

product (Stumpe et al., 2008 ). Changes in the traditional method process

for smoking meat products will be needed to ensure healthy Traditional meat product smoking is a non-optimised food

products. More scientiﬁc background and data are needed to processing technology. The temperature and moisture parameters

elucidate the precise inﬂuence of the use of different types of of traditional smoking chambers depend on the unpredictable,

smoking methods and other process parameters in food contamina- variable values of weather conditions. The usual smoking time for

tion with PAH carcinogenic compounds. traditionally manufactured Spanish chorizo ranges between 10 and

15 days of smoking, the whole process requiring about 3 weeks.

During this process, wood combustion and ﬁre are produced, which

require a temperature above 700 C (Demirbas, 2009 ). The amount

Table 2 of PAH in smoke, formed during pyrolysis, increases linearly with

Benzo(a)pyrene (BaP) content (mg/kg) and multi-range statistical analysis of smoking temperature within the interval 400–1000 C (To ´ th and

commercial chorizo samples (n = 3).

Blaas, 1972 ). Likewise, the PAH content in smoked meat products

Sample BaP (mg/kg) Moisture Multi-range generally increases during smoking (Djinovic et al., 2008 ). This

mean Æ uncertainty (%) statistical analysis: process is not environmentally friendly, as an unnecessary amount

heterogeneous

of excess wood is used for combustion in some cases. In fact, the

groups of BaP content

amount of raw material used to produce smoke is not monitored.

samples (p < 0.05)

Furthermore, safety in the workplace is currently one of the

M 3.03 Æ 0.01 30.73 Group 1

most important factors of industrial quality. Safety in the meat

O 3.21 Æ 0.12 15.13

industry is threatened by traditional uncontrolled smoking

P 2.8 Æ 0.25 24.54 Group 2

C 2.46 Æ 0.15 21.17 Group 3 processes. For instance, over the last 5 years more than 9 smoked

N 2.59 Æ 0.05 20.84 meat product companies located within the geographical scope of

F 1.83 Æ 0.05 18.11 Group 4

this study have burnt down because of uncontrolled combustion of

J 1.46 Æ 0.11 31.45 Group 5

wood. Moreover, product weight losses are not always the same

K 1.46 Æ 0.23 33.22

and product standardisation and homogenisation, nowadays

I 1.27 Æ 0.21 34.65 Group 6

J 1.46 Æ 0.11 31.45 demanded by customers as well as wholesalers, are yet to be

G 1.16 Æ 0.05 38.74 Group 7

achieved in this northern region of Spain. All these facts indicate

I 1.27 Æ 0.21 34.65

that the traditional smoking process needs improving.

D 1.03 Æ 0.04 21.93 Group 8

G 1.16 Æ 0.05 38.74

D 1.03 Æ 0.04 21.93 Group 9

4. Conclusions

L 0.9 Æ 0.05 23.04

H 0.38 Æ 0.08 35.31 Group 10

A 0.41 0.00 16.04 The combination of sonication, SPE and GC/MS was optimised

B 0.52 Æ 0.04 15.68

to reveal new data on the BaP content of 16 smoked meat products

E 0.54 Æ 0.09 36.25

(chorizos) from different producers of an as-yet unstudied region,

94 E. Ledesma et al. / Journal of Food Composition and Analysis 37 (2015) 87–94

Fretheim, K., 1976. Carcinogenic polycyclic hydrocarbons in Norwegian smoked

the Principality of Asturias, linking it with their moisture content

meat sausages. Journal of Agricultural and Food Chemistry 24 (5), 976–979.

to verify the quality of the manufacturing process.

Gilbert, J., 1994. The fate of environmental contaminants in the food chain. The

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Gomaa, E.A., Gray, J.I., Rabie, S., Lopez-Bote, C., Booren, A.M., 1993. Polycyclic

was 0.38–3.21 mg/kg. Five of the 16 direct smoked samples do not

aromatic hydrocarbons in smoked food products and commerical liquid smoke

meet the 2 ppb BaP limit stated by EU regulation No. 835/2011,

ﬂavourings. Food Additives & Contaminants 10, 503–521.

which was introduced on 1/09/2014. The average BaP content Iba´n˜ez, R., Agudo, A., Berenguer, A., Jakszyn, P., Tormo, M.J., Sanche´z, M.J., 2005.

Dietary intake of polycyclic aromatic hydrocarbons in a Spanish population.

found in the samples (1.57 mg/kg) falls below the new permissible

Journal of Food Protection 68, 2190–2195.

BaP limit. The range in moisture content found in the samples was

Karl, H., Leinemann, M., 1996. Determination of polycyclic aromatic hydrocarbons

15.1–38.7%. There was no correlation between BaP concentration in smoked ﬁshery products from different smoking kilns. Zeitschrift fuer

and moisture content. Lebensmittel-Untersuchung und – Forschung 202, 458–464.

Larsson, B.K., Pyysalo, H., Sauri, M., 1988. Class separation of mutagenic polycyclic

Uncontrolled traditional direct smoking methods do not consti-

organic material in grilled and smoked foods. Zeitschrift fuer Lebensmittel-

tute an environmentally friendly, safe or optimised food processing

Untersuchung und – Forschung 187 (6), 546–551.

technology, as they do not allow product standardisation. However, Lodovici, M., Dolara, P., Casalini, C., Ciappellano, S., Testolin, G., 1995. Polycyclic

aromatic hydrocarbon contamination in the Italian diet. Food Additives and

the direct smoking process can be adapted to obtain healthy, good

Contaminants 12, 703–713.

quality products with low moisture content which present a good

Lorenzo, J.M., Purrinõs, L., Bermudez, R., Cobas, N., Figueiredo, M., Garcıá Fontań,

microbiological status and low BaP content. M.C., 2011. Polycyclic aromatic hydrocarbons (PAHs) in two Spanish traditional

smoked sausage varieties: ‘‘Chorizo gallego’’ and ‘‘Chorizo de cebolla’’. Meat

Science 89, 105–109.

Acknowledgements

Martin, D., Ruiz, J., 2007. Analysis of polycyclic aromatic hydrocarbons in solid

matrixes by solid-phase microextraction coupled to a direct extraction device.

Talanta 71, 751–757.

We gratefully acknowledge the support of the Centre for the

Martorell, I., Perello´ , G., Martı´-Cid, R., Castell, V., Llobet, J.M., Domingo, J.L., 2010.

Development of Industrial Technology (CDTI) and the Foundation

Polycyclic aromatic hydrocarbons (PAH) in foods and estimated PAH intake by

for the Promotion of Scientiﬁc and Applied Research and the population of Catalonia, Spain: temporal trend. Environment International

36, 424–432.

Technology in Asturias (FICYT). This work has been funded under

McGrath, T.E., Wooten, J.B., Geoffrey Chan, W., Hajaligol, M.R., 2007. Formation of

the R&D projects: (CDTI funded project no. IDI 20091089) and

polycyclic aromatic hydrocarbons from tobacco: the link between low temper-

(FICYT funded project no. IE07-161). ature residual solid (char) and PAH formation. Food and Chemical Toxicology

45, 1039–1050.

Moret, S., Conte, L.S., 2002. A rapid method for polycyclic aromatic hydrocarbon

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4.2 MECANISMO DE PENETRACIÓN DE BENZO(A)PIRENO EN CHORIZO: INFLUENCIA DEL TIEMPO DE AHUMADO Y LA PROFUNDIDAD

El Código (CAC/RCP 68/2009) del Códex Alimentarius establece las 10 variables que han de ser controladas para reducir la contaminación por hidrocarburos aromáticos policíclicos (HAP) en los alimentos producidos por procedimientos de ahumado y secado directo. Entre estas variables se encuentran el tipo de combustible, el método de ahumado o secado (directo o indirecto), el procedimiento de generación de humo, el uso de humo líquido, la distancia entre el alimento y la fuente de calor, la posición del alimento con respecto a la fuente de calor, el contenido de grasa del alimento y lo que le sucede durante el procedimiento, la duración del procedimiento de ahumado o secado directo, la temperatura y la limpieza y el mantenimiento de los utensilios. Cada una de estas variables ha sido estudiada por varios autores, concluyendo que en muchos casos, en especial los incontrolados sistemas tradicionales de ahumado a modo directo, es difícil controlar todas las variables, por lo que deben estudiarse por separado. Por otro lado, como tratamiento posterior al ahumado el código aconseja medidas para eliminar el hollín y las partículas que contienen HAP en la superficie del alimento.

En el presente trabajo se presentan los resultados del estudio de una de estas variables, el tiempo de ahumado, en el proceso de elaboración de chorizo asturiano a modo directo. Así mismo, por primera vez se realiza un estudio pormenorizado de la penetración del marcador benzo(a)pireno y evacuación de la humedad en distintas profundidades de chorizo ahumado. Se realiza especial hincapié en evaluar el proceso de concentración del producto durante el secado. Con los resultados obtenidos se propone por primera vez un mecanismo que define el proceso de contaminación del chorizo por HAP durante el ahumado, ofreciendo datos relevantes para la comunidad científica.

Publicación: Ledesma, E., Rendueles, M., & Díaz, M. (2014). Benzo(a)pyrene penetration on a smoked meat product during smoking time. Food Additives and Contaminants, 31, 1688-1698.

Situación: Artículo publicado en Food Additives & Contaminants: Part A (Elsevier).

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Food Additives & Contaminants: Part A Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/tfac20 Benzo(a)pyrene penetration on a smoked meat product during smoking time E. Ledesma ab , M. Rendueles a & M. Díaza a Department of Chemical Engineering and Environmental Technology, University of Oviedo, Oviedo, Spain b Research, Development and Innovation (R&D+i) Department, El Hórreo Healthy Food S.L, Noreña, Spain Accepted author version posted online: 31 Jul 2014.Published online: 22 Aug 2014.

To cite this article: E. Ledesma, M. Rendueles & M. Díaz (2014): Benzo(a)pyrene penetration on a smoked meat product during smoking time, Food Additives & Contaminants: Part A, DOI: 10.1080/19440049.2014.949875

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Benzo(a)pyrene penetration on a smoked meat product during smoking time E. Ledesma a,b, M. Rendueles a and M. Díaz a* aDepartment of Chemical Engineering and Environmental Technology, University of Oviedo, Oviedo, Spain; bResearch, Development and Innovation (R&D+i) Department, El Hórreo Healthy Food S.L, Noreña, Spain (Received 19 May 2014; accepted 22 July 2014 )

The Codex Alimentarius gives recommendations to prevent carcinogenic polycyclic aromatic hydrocarbons (PAH) (repre- sented by benzo(a)pyrene (BaP)) contamination during processing of meat products, including the control of smoking time. The in ﬂuence of direct smoking time (0, 1, 3, 5 and 7 days) on the relationship between the BaP and moisture content of a typical Spanish smoked meat product called chorizo and the mechanism of BaP penetration and water release from four different depths in the product was studied. Chorizo was studied from the Principality of Asturias, a location never before tested. An analytical method was developed for this purpose consisting of PAH extraction assisted by sonication followed by solid-phase extraction (SPE) sample clean-up and analytical determination using GC-MS. Results show that an increase in smoking time produced contradictory and independent effects on the moisture and BaP content (µg kg –1) of chorizos. The moisture content decreased from 49.9% to 31.3%. On the other hand, the BaP content increased from less than 0.24 µg kg –1 to 0.75 µg kg –1, ﬁnally stabilising after 5 days of smoking. After this time, the natural pores of the casing could be blocked by the large size tar particles from smoke, preventing the continued penetration of PAHs. The BaP content decreased and the moisture content increased progressively from the casing to the centre of the meat product. BaP mainly accumulated in the smoked casing, being four times in excess of the legal limit. This paper analyses the mechanism for preventing PAHs contamination during the process of smoking meat products. Keywords: benzo(a)pyrene (BaP); moisture content; smoking time; depth; polycyclic aromatic hydrocarbons (PAHs); smoked meat product

Introduction structures. Above 750ºC, tertiary tar products begin to Meat and meat products are high-value foods. Meat pro- appear with increasing temperature. Condensed tertiary duction is foreseen to double by 2050 due to its nutritional aromatics make up a series of polynuclear aromatic hydro- composition, essential for the growing world population. carbons (PAHs) without substituents (atoms or a group of Meat products have high-quality protein, containing all the atoms substituted by hydrogen in the parent hydrocarbon essential amino acids, and highly bioavailable minerals chain) (Basu 2010). and vitamins (Food and Agriculture Organization of PAHs have been found to be carcinogenic, mutagenic, fi United Nations 2013). lipophilic and bioaccumulative. The most signi cant endpoint Meat products have been smoked since the beginning of PAH toxicity is cancer (ATSDR 1995 , 2009 ). Benzo(a) of time, principally for the purpose of food preservation pyrene (BaP) is the current marker for the occurrence and Downloaded by [Manuel Rendueles] at 11:04 26 August 2014 via drying (Möhler 1978; Varlet et al. 2007). Smoking effect of PAHs in meat products according to European Union technology continues to be used nowadays because of Commission (EC) Regulations No 1881/2006 and (EU) No fi the sensory active components contained in smoke (phe- 835/2011. This new regulation speci es that two dates must nol derivates, carbonyls, organic acids and their esters, be considered as regards PAH contamination in foodstuffs. lactones, pyrazines, pyrols and furan derivatives) in the New maximum levels for the sum of four substances (PAH4) fl aromatisation and colouring of meat products, widely (BaP, benzo(a)anthracene, benzo(b) uoranthene and chry- demanded characteristics by the consumer ( Šimko 2002) sene) were introduced, maintaining a separate maximum and due to the antioxidant action of certain components, level for BaP, to ensure comparability between previous and especially phenol derivatives (Möhler 1978). future data. Moreover, a recent study by Lorenzo et al. ( 2011 ) Food can be contaminated during smoking by unde- reports it to be a good marker for the sum of 15 PAHs as well sired by-products formed via biomass gasi fication and as for seven PAHs considered probable human carcinogenic ‘ ’ pyrolysis, namely tar. Tar is a thick, black and highly by the USEPA in chorizo gallego . viscous liquid that can create the following problems: Humans are exposed to PAHs due to environmental direct condensation and deposition on foods, formation contamination, though mainly because of diet (Lodovici of tar aerosols and polymerisation into more complex et al. 1995; Phillips 1999; Falcó et al. 2003; Ibáñez et al.

*Corresponding author. Email: [email protected]

2005 ). Food can be PAHs contaminated because of envir- Böhnke 1975 ; Fretheim 1976 ; Chiu et al. 1997 ; Šimko onment contamination and manufacturing processes such 2002 ). Modern methods include microwave-assisted extrac- as smoking, drying, roasting, baking, barbecuing and fry- tion (MAE), sonication (Purcaro et al. 2009 ), accelerated ing (Rey-Salgueiro et al. 2008 , 2009; Rey-Salgueiro, solvent extraction (ASE) (Djinovic et al. 2008 ; Martorell García-Falcón, et al. 2008; Rey-Salgueiro, Martínez- et al. 2010 ; Sun et al. 2012 ), ultrasound-assisted solvent Carballo, et al. 2008 Codex Alimentarius Commission, extraction (USAE), ultrasound-assisted emulsi fication – CAC/RCP 68-2009). In non-occupational settings, up to microextraction (USAEME) (Yebra-Pimentel et al. 2013 ) 70% of PAH exposure for a non-smoker can be associated or pressurised liquid extraction (PLE) (Pöhlmann et al. with the diet (Skupinska et al. 2004). Although several 2013 ), followed by SPE (García-Falcón et al. 2004 , 2005 ; foodstuffs are contaminated with PAHs, high levels have Purcaro et al. 2009 ; Hitzel et al. 2013 ), stir-bar sorptive been found in particular in smoked meat products, thus extraction (SBSE) (García-Falcón et al. 2004 ) or solid- making their study a matter of importance (Larsson et al. phase microextraction (SPME). Direct extraction devices 1988 ; Gomaa et al. 1993; Karl & Leinemann 1996; (DED) have also been proposed (Martin & Ruiz 2007 ). Martorell et al. 2010). The combination of sonication with SPE methods has been To protect consumers from these contaminants, the tested in some foodstuffs, giving results that are both fast and European Commission has set maximum levels for PAHs inexpensive (Purcaro et al. 2009 ). Typical analytical methods in foodstuffs. The current maximum permissible BaP content for PAH identi fication are: HPLC combined with fluores- – in smoked meat and smoked meat products is 5.0 μg kg 1 cence (FLD) or ultraviolet (UV) detectors (Moret & Conte – (wet weight) until 31 August 2014 and will be 2.0 μg kg 1 2002 ; Purcaro et al. 2009 ; Lorenzo et al. 2011 ; Santos et al. (wet weight) from 1 September 2014 on (European Union 2011 ; Roseiro et al. 2012 ) and GC combined with flam e Commission (EU) Regulation No. 835/2011). The permissi- ionisation (FID), ion trap (ITD) and mass spectrometry ble sum of PAH4 in these foods will be 30.0 μg kg –1 in wet (MSD) detectors (Hitzel et al. 2013 ; Olatunji et al. 2014 ). weight from 1 September 2012 to 31 August 2014 and GC-MS and GC/HRMS are widely used nowadays to deter- 12.0 μg kg –1 in wet weight from 1 September 2014 on. mine PAHs in foodstuffs ( Šimko 2002 ; Martin & Ruiz 2007 ; The USFDA regulates contaminant levels in foodstuffs, but Purcaro et al. 2007 ; Djinovic et al. 2008 ; Stumpe-V īksna has not still established standards governing the PAH content et al. 2008 ; Martorell et al. 2010 ; Wretling et al. 2010 ; Hitzel of foodstuffs. The maximum contaminant level goal for BaP et al. 2013 ). in drinking water is 0.2 parts per billion (ppb). In 1980, the The main aim of the present study was to determine the USEPA established ambient water quality criteria to protect in fluence and relationship of direct smoking time on the BaP human health from the carcinogenic effects of PAH expo- and moisture content of a traditional Spanish meat product sure. The recommendation was a goal of zero (non-detect- called chorizo in an as-yet unstudied region, the Principality able level for carcinogenic PAHs in ambient water). As a of Asturias, to assess the quality of drying with regard to BaP regulatory agency, the USEPA establishes a maximum con- content, according to European Union Commission (EU) taminant level (MCL) for BaP, the most carcinogenic PAHs, Regulation No. 835/2011. Moreover, the BaP and moisture at 0.2 ppb (ATSDR 2009; EPA 2013). content at different depths in chorizo were assessed in order The ‘Code of Practice for the Reduction of to de fine the mechanism of penetration of this compound Contamination of Food with PAHs from Smoking and into the food product during drying. An analytical method ’ Downloaded by [Manuel Rendueles] at 11:04 26 August 2014 Direct Drying Processes (CAC/RCP 68/2009) and recent based on a combination of sonication, SPE and GC-MS was studies (Djinovic et al. 2008 ; Stumpe-V īksna et al. 2008 ; developed and employed for this purpose. Lorenzo et al. 2011 ; Santos et al. 2011 ; Roseiro et al. 2012 ) state that PAH contamination in foodstuffs via food processing should be controlled by taking into consideration sev- Materials and methods eral variables of food processing technology. One of these Sample preparation parameters is the duration of the smoking process. They All samples comprise the same kind of typical Spanish especially specify that smoking time should be as short as smoked meat product called chorizo, made of the following possible to minimise the exposure of food surfaces to PAH- ingredients: pork loin (46.8%), pork jowl (46.8%), salt bearing smoke, but long enough to allow the product to be (1.8%), garlic (1%), sweet or spicy paprika (2%) and thoroughly cooked and to ensure food safety, shelf life, and herbs (1.6%), minced, mixed and encased in a natural good sensory and nutritional properties. casing made from animal intestine. Antioxidants such as A variety of both classical and modern pre-treatment and sodium citrate or sodium ascorbate, colourings such as analytical methods have been used to determine PAHs in carmine, emulsi fiers such as sodium triphosphate, food foods. Classical pre-treatment methods include saponi fica- preservatives such as sodium nitrite and other food addition, liquid –liquid extraction and Soxhlet in combination tives were used to ensure the microbiological stability and with gel permeation chromatography (GPC) (Grimmer & quality of the product. Food Additives & Contaminants: Part A 3

All samples were manufactured at the El Hórreo Healthy Food S.L. meat company facilities, where 50 kg of chorizo raw materials were minced, mixed, marinated and stuffed to obtain 200 chorizo strings of four chorizos. Then chorizos were exposed to traditional direct smoking. A mixture of oak (90%) and chestnut (10%) woods was used for smoking. All the selected samples were placed in the same position in the smoking chamber, at the same distance from the smoke source, namely 10 m. The nutritional details of a 5-day smoked chorizo manufactured by the company with the same ingredients and manufacturing process like the one studied here, determined by the Principality of Asturias Meat Industry Association Technological Centre for Supporting Innovation (Spanish acronym, ASINCAR), are: 31.41% Figure 1. (colour online) Sample dimensions employed in the moisture content, 22.98% protein, 37.94% fat, 2.85% car- study on BaP content at different chorizo depths. bohydrates, 4.82% ash and pH 5.2. The samples were classi fied according to the two kinds Table 1. Chorizo dimensions and mass for depth study. of studies carried out: those made to study the in fluence of smoking time on the BaP and moisture content of chorizo; Dimension Length (cm) Depth/sample Mass (g) Mass (%) and those made to study the moisture content and penetration of BaP at different depths of smoked chorizos. L1 13.7 D1 1.15 1.00 L2 3.75 D2 23.6 20.5 L3 3.25 D3 65.4 56.8 L4 0.50 D4 25.0 21.7 Study on the BaP content in chorizo during smoking time Total 115.1 100.0 One string of four chorizos was selected before the smoking process. This sample was denominated the ‘t0 sample ’. Four strings of four chorizos were hung 40 cm apart on a (D4). The amount of chorizo located between D4 and D2 bar, placed in the same position and location in the smoking was denominated depth 3 (D3). Figure 1 and Table 1 show chamber and collected after the following smoking times: 1, the average dimensions and weights of depths of the 3, 5 and 7 days. These samples were respectively denomi- chorizos de fined in this study. nated samples t1, t3, t5 and t7. Once collected, the samples were labelled and stored in the dark at 18ºC. Reagents and standards Benzo(a)pyrene (BaP) analytical standard solution was Study on the BaP content at different depths of chorizo supplied by Sigma-Aldrich (Seelze, Germany) at a con- – One string of four chorizos was hung on a bar, placed in centration of 100 µg ml 1 in cyclohexane and a total

Downloaded by [Manuel Rendueles] at 11:04 26 August 2014 the smoking chamber and collected after 5 days of smok- volume of 2 ml. ing. Once collected, the samples were labelled and stored BaP internal standard (D12, 98%) solution was in the dark at 18ºC. Two chorizos from the string were obtained from Cambridge Isotope Laboratories (Andover, – chosen for the experiments. The chorizos were denomi- MA, USA) at a concentration of 200 µg ml 1 in isooctane nated samples A and B, respectively. The overall length and a total volume of 1.2 ml. and width of the samples were 12.5 and 4 cm for sample All solvents for sample preparation, hexane, dichlor- A and 15.0 and 3.5 cm for sample B, respectively. The omethane, cyclohexane and isooctane (2,2,4-trimethylpen- overall chorizo weights were 116.9 and 113.4 g, respec- tane) were purchased from Sigma-Aldrich, puriss grade, tively, for samples A and B. During the sampling process, ACS reagent, ≥ 99% (GC). four depths were de fined for each chorizo: D1, D2, D3 SPE cartridges were supplied by Varian (Palo Alto, and D4. The casing of the sample was denominated depth CA, USA) and Agilent Technologies (Santa Clara, CA, 1 (D1). Once the casings were removed, samples A and B USA). Mega Bond Elut cartridges contain a silica sorbent were cut in slices to obtain the amount of product de fining phase, with a particle size of 40 nm, a weight of 5 g and a the following depths: the amount of chorizo situated at a volume of 20 ml. distance between 0.25 cm from the casing and the casing Amber-coloured flasks and vials were used to protect of the chorizo was denominated depth 2 (D2). The amount the PAHs from light decomposition. In particular, 15 ml of chorizo situated at a distance of 0.25 cm from the centre (21 × 70 mm) amber glass vials were purchased from and the centre of the chorizo was denominated depth 4 Sigma-Aldrich for PAHs collection during the SPE step. 4 E. Ledesma et al .

Sample pre-treatment procedure in a Supelco Visiprep TM SPE vacuum mani- Grinding step fold with 12 ports (Sigma-Aldrich), was used to isolate BaP and BaP-d12 (the sought after PAHs in the sample) First, the casing of the chorizo was removed and stored. similar to the previously designed set-up for rapid PAHs Then, 200 g of chorizo for the A experiments and the total determination in vegetable oils by Moret & Conte ( 2002). amount of each depth for the B experiments were ﬁnely First, a 5 g silica SPE cartridge (Mega Bond Elut, 20 homogenised in a meat grinder (Minirobot D81 meat ml, Varian) was slowly washed with 20 ml of dichloro- grinder, Moulinex, France) following the protocol devised methane, dried completely under vacuum and conditioned by Purcaro et al. ( 2009). Pre-treatment of all samples was with 20 ml of n-hexane (to prevent the cartridge from carried out in triplicate. drying). Next, 1 ml of the sonicated sample was diluted in 3 ml of n-hexane and slowly loaded into the cartridge, Lyophilisation step preventing the cartridge from drying completely. The aliphatic hydrocarbons were then discharged via the addition Three aliquots of 20 g chorizo, or the total amount in the of 8 ml of a mixture of hexane and dichloromethane 70:30 case of the D1 samples, were taken, placed in individual (v/v) to the cartridge. Finally, after loading the cartridge lyophilisation plates, carefully sealed with aluminium foil with another 8 ml of the hexane and dichloromethane and weighed before freeze-drying. Each sample was then 70:30 (v/v) solution, the PAHs, BaP and BaP-d12 con- – frozen to 80ºC for 4 h and freeze-dried in a lyophiliser tained in the sample were slowly discharged into 15 ml (Telstar-Cryodos, Spain) for 1 day. Finally, the plates amber glass vials, allowing the cartridge to be dried under (containing the sample) were weighed after freeze-drying. vacuum.

Sonication step A total of 2 g of freeze-dried chorizo (one from each lyophi- Concentration step lisation plate), or the total amount in the case of the D1 The samples were concentrated after the SPE step. The samples after pulverisation, were placed in flasks (the remain- volume being made up to 1 ml by N 2 blowing using a ing amount of lyophilised chorizo was stored in a freezer). nitrogen stream at the top of the 15 ml vials placed in a Then, 20 ml of n-hexane and 200 µl of benzo(a)pyrene-d12 fume hood. (BaP-d12) internal standard solution at a concentration of Finally, the samples were loaded into 2 ml GC-MS 100 µg l –1 were added (dilutions from the stock solution amber glass vials and the final volume of 1 ml was must be made previously). To help the deuterated compound checked by weighing the vials. adapt to the sample matrix, this solution was carefully stored under refrigeration for 3 days. This is the appropriate point to add the internal standard compound as it is the first time both compounds are in the same matrix (Stumpe-V īksna et al. Analysis 2008 ; Purcaro et al. 2009 ). Finally, the samples were soni- GC-MS calibration cated for 1 h in an ultrasound bath (Cod. 3000513, J.P. BaP quantification was performed via prior calibration of Selecta, S.A, Barcelona, Spain) at ambient temperature.

Downloaded by [Manuel Rendueles] at 11:04 26 August 2014 the GC-MS system with BaP and BaP-d12 standard solutions. For calibration purposes, BaP-d12 standard solution was added to check the extraction recovery during pre- Filtration treatment methods. After sonication, the samples were filtered on a paper filter Calibration curves were generated by injecting six (reference number 1242, 11 cm, Albet LabScience; Albet- diluted standard solutions of BaP and BaP-d12 in cyclo- Hahnemuehle S.L, Barcelona, Spain)) to remove the solid hexane, in triplicate. BaP standard concentrations for each meat waste, which was washed with additional n-hexane calibration vial were: 0.05, 0.1, 0.5, 1.0, 5 and 10 ppb. The (5 + 5 ml). The solvent was subsequently collected in a concentration of BaP-d12 for all calibration vials was 2 flask, making up the volume to 10 ml with the help of a ppb. The MS assistant program (Agilent Technologies) rotary evaporator (Heidolph Laborota 4000 ef ficient, compares the relationship between both BaP standard Heidolph-instruments, Schwabach, Germany) and the signals (BaP and BaP-d12) to determine the final BaP addition of the necessary amount of n-hexane. concentration. Identi fication of compound was assessed by comparing the retention time and con firm the ion ratios (qualifier ion/target ions) with a criterion of ±20% of SPE step standards compounds ratio. The criterion of BaP/BaP- The extract obtained after sonication contained an appre- d12 standards target ion ratios was ±20%. A blank (i.e. a ciable amount of fat (Purcaro et al. 2009 ). An SPE vial containing only the solvent cyclohexane, with no BaP Food Additives & Contaminants: Part A 5

or BaP-d12 standard compounds) was also included LODs and LOQs were de fined as the concentration of among all the standard vials containing BaP compounds. the analyte that produced a signal-to-noise ratio of 3 and 10, respectively. Moreover these results were con firmed by the analysis of blank samples and six diluted standard GC solutions of BaP and BaP-d12 in cyclohexane, in tripli- Separation of BaP and BaP-d12 was performed on an cate. The slope (m) and standard deviation of intercept HP-5MS 19091S-433 part number column (sb 0) of analytical response ( y) versus concentration level (30 m × 250 μm × 0.25 μm) in a 6890 gas chromatograph added (x) were calculated, then LOD and LOQ were also (GC) (all Agilent Technologies). The injection temperature estimated with following equations: LOD = 3.3* sb 0/ m and volume were 300ºC and 1 µl (splitless), respectively. and LOQ = 10* sb 0/ m. Helium was applied as carrier gas at a constant flow rate of 1.5 ml min –1. The temperature ramp used was the following: isothermal at 55ºC for 1 min, increasing at a rate of Statistical analysis 25 °C min –1 to 320ºC for 3 min, total run time 14.60 min. Data from each study were analysed by running Fisher ’s least significant difference (LSD) procedure for multi-sample statistical comparison. This method was used to dis- MS criminate among the means. Besides t-tests between- Identification of BaP and BaP-d12 was performed using consecutive samples were applied to compare means at a an Agilent Technologies 5975 mass spectrometer detector 5% probability level. Previously F-tests at a 5% probabil- (MS) equipped with a G2589-20045 part number 6-mm ity level were carried out to compare SDs. Standardised ultra-large aperture draw-out lens. The solvent delay was skewness and standardised kurtosis were used to assess if 4.00 min and the EM voltage (run at auto-tune voltage) the samples came from normal distributions. The software was 1294 V. The low and high masses scanned were 252 used was STATGRAPHICS Plus 3.1. and 264 amu, respectively. SIM mode was selected for one group, three ions per group (252, 264 and 126), 50 ms dwell/ion. The quad temperature was 180°C, the source Results and discussion temperature 300°C and the transfer line temperature 280° All samples were successfully analysed by the proposed C. Data were acquired by operating the MS in ion-mon- method. Previous studies consisting of the combination of itoring mode. Peak spectra were compared with the mass Soxhlet and gel permeation chromatography (GPC) were spectra of BaP and BaP-d12 standards and the library carried out in order to evaluate analytical methods for BaP supplied with the instrument. Nine different spiked sam- determination in smoked meat products. The combination ples (as well as unspiked controls) were prepared and each of the sonication and SPE pre-treatment methods to deter- sample and vial were analysed in triplicate according to mine the BaP content in smoked meat products by GC-MS the analytical procedure. Recoveries were calculated by analysis was found to be a good method, as it entails a low comparing the difference between spiked and unspiked level of solvent consumption and waste generation, short samples with the known amount of added BaP standard operating times and is easy to set up. Figure 2 shows the (1 ppb). LODs and LOQs were evaluated on the basis of BaP and BaP-d12 target ions chromatograms of a casing

Downloaded by [Manuel Rendueles] at 11:04 26 August 2014 noise obtained with the analysis of the blank samples. chorizo sample obtained by SIM analysis mode of

Figure 2. BaP (mass 252 m/z, black upper chromatogram) and BaP-d12 internal standard (mass 264 m/z, grey lower chromatogram) target ions chromatograms of casing sample. 6 E. Ledesma et al .

GC/MS. Similar retention time was found for BaP and The BaP content between the samples smoked during BaP-d12 standards, 12.47 and 12.49 s respectively. Ion 3 and 5 days shows a statistically signi ficant difference ratios in all samples were within the required criterion (p < 0.05). However, no signi ficant differences (p > 0.05) (±20) and compounds were con firmed. This method offers were observed between the BaP content of samples high-quality quantitative results with high extraction ef fi- smoked during 5 and 7 days. Fisher ’s least signi ficant ciency. The LOD found was 0.05 µg kg –1 and the LOQ difference (LSD) procedure found two groups, one for found was 0.24 µg kg –1. Recoveries ranged from 99% to the meat products smoked for 3 days and another group 102% and RSDs were lower than 4%. for the products smoked for 5 and 7 days. Finally, it can be stated that BaP becomes stabilised after 5 days of smoking. The differences between moisture contents between In fluence of direct smoking process time on the BaP and the samples was similar to the BaP content. Signi ficant moisture content of chorizo differences (p < 0.05) were observed between samples All the samples were analysed following the proposed smoked for 1 and 3 days and for 3 and 5 days, respec- method. Figure 3 shows the moisture and BaP content tively. No signi ficant differences were observed between (µg kg –1) found in the five analysed chorizos processed the samples smoked for 5 and 7 days ( p > 0.05). – with different smoking times (0, 1, 3, 5 and 7 days). As The BaP content (µg kg 1) results in the freeze-dried Figure 3 shows, an increase in smoking time produces samples compared with fresh samples shown in Figure 4 opposite effects on the moisture (%) and the BaP content which show that the increase in BaP content is not only (µg kg –1) of chorizo. While the moisture content decreases caused by the decrease in moisture content in the direct (49.9% ± 3.2%, 42.9% ± 2.5%, 36.9% ± 0.4%, smoked product, as the BaP content of the samples 33.3% ± 1.0% and 31.3% ± 1.2%), the BaP content expressed in this way also increases with smoking time. increases and finally becoming stabilised after 5 days of Visual inspection of the samples revealed that the stabili- smoking (from less than 0.24 µg kg –1 LOQ for 0 and 1 sation of the BaP content after 5 days of smoking may be days of smoking to 0.37 ± 0.05, 0.75 ± 0.05 and produced by obstruction of the casing pores. 0.75 ± 0.05 for 3, 5 and 7 days of smoking respectively). These results are in agreement with those obtained by Moreover, the intensity of the effects is also different. In Djinovic et al. ( 2008) who found that the BaP and total the same smoking time (7 days), while the moisture con- PAHs content in the products they studied generally tent decreased 18.7% (from 49.9% ± 3.2% to increased during smoking. However, these authors found 31.3% ± 1.2%), the BaP content increased more than only a similar BaP content in smoked meat products, – 300% (from less than 0.24 µg kg –1 LOQ to always less than 0.48 µg kg 1 after 9 days of smoking, – 0.75 ± 0.05 µg kg –1). The BaP content of chorizo was whereas we found 0.75 µg kg 1 after 5 days of smoking. doubled from 3 days of smoking (0.37 ± 0.05 µg kg –1) to Santos et al. ( 2011) studied the BaP content of a chorizo 5 days of smoking (0.75 ± 0.05 µg kg –1). with similar characteristics to those studied in this paper, Downloaded by [Manuel Rendueles] at 11:04 26 August 2014

Figure 3. (colour online) In ﬂuence of direct smoking time (0, 1, 3, 5 and 7 days; n = 3) on the moisture (%) and BaP ( μg kg –1) content of a direct smoked meat product (chorizo) analysed without casing (D1). Food Additives & Contaminants: Part A 7

or smoking time (Djinovic et al. 2008; Stumpe-V īksna et al. 2008, Codex Alimentarius, CAC/RCP 68-2009, Lorenzo et al. 2011; Santos et al. 2011; Roseiro et al. 2012; Hitzel et al. 2013).

BaP penetration at different depths in a meat product during the direct smoking process In this study the BaP (µg kg –1) and moisture (%) content at four different depths of two meat products (chorizos A and B) smoked in the same conditions (smoking time, smoking room, distance to the smoking source, ingredients, etc.) were analysed in triplicate. The results obtained were similar for the two studied meat products, so an average of both has been used in the discussion of the results. Figure 5 shows the moisture (%) and BaP content (µg kg –1) found at the four studied depths. As can be appreciated in Figure 5, several differences were found in the BaP content at the studied depths. There are impor- Figure 4. (colour online) BaP content in freeze dried and fresh tant differences in the moisture and BaP content found at chorizo during smoking time (0, 1, 3, 5 and 7 days; n = 3). depth D1 (casing) compared with the rest of the studied depths. The BaP content found in the samples decreases from the casing towards the inside of the meat product. with 25.1% fat content, 5 days of smoking and 2 cm in The BaP content found in the casing (D1, diameter. These authors reported a BaP content of –1 –1 20.0 ± 1.1 µg kg ) is very high, while the BaP contents 0.38 µg kg in this chorizo, nearly the same BaP content found inside the chorizo are similar and low we found after 3 days of smoking. After 5 days of smok- –1 –1 –1 (1.63 ± 0.11 µg kg D2, 1.12 ± 0.10 µg kg D3 and ing, we found twice the content of BaP, 0.75 µg kg . 0.88 ± 0.10 µg kg –1 D4), with concentrations decreasing These results may be considered similar bearing in mind with depth. It is important to note that signi ﬁcant differ- ﬂ the great number of parameters that in uence the tradi- ences between the samples (p < 0.05) were obtained by tional direct smoking method, such as the kind of wood running t-tests (D2 –D3, D3 –D4). Three different groups used for combustion, the position in the smoking chamber were found by running Fisher ’s LSD procedure of D2, D3 Downloaded by [Manuel Rendueles] at 11:04 26 August 2014

Figure 5. (colour online) BaP ( μg kg –1) and moisture (%) content at different depths (D1, D2, D3 and D4) in a 5 days ’ direct smoked meat product (chorizo). 8 E. Ledesma et al .

and D4 samples. Figure 2 shows the BaP and BaP-d12 moisture content found in the casing (D1, ions chromatogram found in the casing. As Figure 2 12.4% ± 1.4%) is very low, while the values found inside shows, the BaP peak size is higher than the added internal the chorizo are similar and higher than that at D1 (D2: standard BaP-d12 one. 29.2% ± 1.5%, D3: 35.5% ± 0.3% and D4: 33.8% ± 1. The results obtained in this study indicate that, during 3%). Significant differences (p < 0.05) between D1 –D2 direct smoking process, the greatest amount of BaP, PAH and D2 –D3 were found. No signi ficant differences content indicator, was deposited in the casing of the meat (p > 0.05) between D3 and D4 were found. product, not inside the product. This is an important find- One of the most important goals of the smoking pro- ing, as the inhabitants of the region where this study was cess during chorizo manufacturing is that of drying. The carried out do not remove the casing of the meat product water content of meat products decreases during drying, before eating it. Chorizo is commonly used as ingredient thus leading to a drop in moisture content. This reduction in traditional Spanish stews such as white beans, lentils or in moisture content is important because it has an inacti- chick peas, as well as in rice dishes such as paella. vating effect of the growth of microorganisms. During the Chorizo casing is not usually removed before preparing traditional direct smoking process using natural casings, these dishes. the product is dried from the outside of the product However, the amount of BaP found inside the chorizo inwards. This process has an effect on the firmness, col- (at depths D2, D3 and D4) is similar to that found in other our, taste and other organoleptic properties of the product. studies. These results are in agreement with those reported The outer layer is harder and more colourful and tasty. by other authors in similar smoked meat products. Garcia- Bearing all this in mind, a scheme of the mechanism Falcón & Simal-Gándara ( 2005), Djinovic et al. ( 2008), of BaP penetration and water exiting the different depths Andrée et al. ( 2010) and Santos et al. ( 2011) report that of a smoked meat product during smoking time is pro- PAHs accumulate on the surface of the smoked meat posed in Figure 6. As summed up, before smoking chorizo product during smoking and then migrate into the pro- is a raw mix of pink ingredients stuffed in a casing without ducts being smoked. Santos et al. ( 2011) speci fically any BaP content. During smoking, chorizo is immersed in found a substantially higher PAHs content in the casing smoke in direct contact with smoke components. After 1 of chorizo than inside the product. day of smoking, smoke deposits start to accumulate on the As is well known, a great number of variables alter the surface of the casing; BaP is mainly deposited in the PAH content of smoked foods, such as the smoking or casing and the first depths of the product start to be BaP drying method (direct or indirect) or the wood used for contaminated and dried. From 1 to 5 days of smoking, combustion (Codex Alimentarius CAC/RCP 68-2009; smoke components (including BaP) continue to accumu- Stumpe-Vīksna et al. 2008 ) and should be speci fied in late mainly on the casing and BaP penetrates deeper inside scienti fic papers. However, the present study shows that the product, while water exits the product from the exter- it is also important to specify whether the samples have nal to the internal depths of chorizo. After about 6 days of been analysed with or without the casing. smoking, smoke components have built a barrier impeding The results obtained in this study could explain the BaP penetration, although in some cases the casing findings of Djinovic et al. ( 2008). They explain that a becomes degraded or torn, thus allowing BaP to penetrate. decrease in BaP content during smoking time could be During this process, water exits the meat product resulting

Downloaded by [Manuel Rendueles] at 11:04 26 August 2014 caused by differences in the content of the smoke during in different depths, being harder and darker on the outside this process. We would further add that the depth of the than inside the product. sample taken for analysis and the obstruction of the pores To sum up, it may be proposed that the sides of the caused by the components of the smoke could also have product, which are in direct contact with the smoke, an in ﬂuence on the results. Moreover, an excessive drying become more dried and PAHs contaminated than those time sometimes produces deterioration or tearing of the that are protected inside the product. The external sides casing, allowing BaP to pass inside the product. This can become obstructed by smoke components, thereby effect could explain an increase in BaP in meat products constituting a barrier to PAHs penetration into the internal smoked over 7 days. Furthermore, we assume that the sides. stabilisation of the BaP content in chorizo after 5 days of smoking may be produced by a coating created by the smoke components on the surface of the product, imped- BaP content in chorizo with regard to regulations ing the penetration of BaP. According to European Union Commission (EU) On the other hand, as Figure 5 shows, there are also Regulation No. 835/2011, in addition to the new PAH4 important differences in the moisture content found at sum, the separate maximum level for BaP is maintained to depth D1 (casing) compared with the rest of the studied ensure comparability of previous and future data. The depths. In this case, the moisture content found inside the recent study by Lorenzo et al. ( 2011) concluded that BaP meat product increases from the casing inwards. The is a good marker for the sum of 15 PAHs in chorizo Food Additives & Contaminants: Part A 9

Figure 6. (colour online) Mechanism of BaP penetration and drying at different depths in a smoked meat product (chorizo) during smoking time.

gallego as well as for seven PAHs considered probable and variable values of temperature and humidity depend- human carcinogens by the USEPA in ‘chorizo gallego ’ ing on weather conditions, the distance between the heat and ‘chorizo de cebolla ’, similar smoked meat products to source and the product, the time of smoking, the amount the one studied here ( ‘chorizo asturiano ’). of fat in the product, the kind of wood, etc., corroborated As Figure 3 shows, the BaP content of a typical chorizo by CAC ( 2009) and reported by several authors (Djinovic – manufactured in 7 days of direct smoking (0.75 μg kg 1) et al. 2008; Stumpe-V īksna et al. 2008; Lorenzo et al. meets the current (5 μg kg –1) and future (2 μg kg –1) legal 2011; Santos et al. 2011; Roseiro et al. 2012), it is quite limits for BaP in smoked meat products. The BaP content difficult to optimise and have full control of traditional increases twofold in 2 days of smoking (from 3 to 5). As direct smoking processes. However, a BaP content below already stated, these samples were analysed without casing. 1 μg kg –1 in similar smoked meat products has been Figure 5 shows that the chorizo depth samples ana- reported by other authors (Jira et al. 2006; Purcaro et al. lysed in this study present different BaP contents. With a 2009; Santos et al. 2011; Roseiro et al. 2012) and also content of 20.0 ± 1.1 µg kg –1, the casing of the smoked below the maximum value allowed by European Union product (D1) presents a value four times in excess of the regulations (5 μg kg –1) (Fontcuberta et al. 2006; Wretling Downloaded by [Manuel Rendueles] at 11:04 26 August 2014 current legal limit set at 5 μg kg –1 (wet weight). However, et al. 2010 ; Lorenzo et al. 2011). In contrast, a BaP the internal depths (D2: 1.63 ± 0.11 µg kg –1, D3: content higher than 2 μg kg –1 has been reported in some 1.12 ± 0.10 µg kg –1 and D4: 0.88 ± 0.10 µg kg –1) kinds of direct smoked chorizo (Roseiro et al. 2012). comfortably meet the current (5 μg kg –1) and future (2 Finally, we can state that the traditional direct smoking μg kg –1) legal limits for BaP in smoked meat products. process is affected by a large number of parameters. A low The total average mass of the studied meat products is BaP content has been found in recent studies of traditional 115.1 4 g and their total average BaP content is 0.16 μg. direct smoked meat products, indicating that it can be This means that the total average BaP content (µg kg –1) in achieved. However, information on innovative smoking the meat products (CT) is 1.36 μg kg –1. If all the studied technologies or the introduction of simple modi fications parts of the meat product were mixed together, a concen- in the smoking process will be necessary in some cases to tration of 1.36 μg kg –1 of BaP would be obtained. On the meet the forthcoming maximum level of BaP allowed in other hand, if the average BaP content of the internal smoked meat products by the European Union regulation, depths (D2, D3 and D4) is studied, a concentration of i.e. 2 μg kg –1. For instance, we propose replacing direct 1.21 μg kg –1 is obtained. Both results will meet the future smoking methods by indirect techniques, such as friction legal limit (2 μg kg –1) for BaP in smoked meat products. smoke generation, particularly controlling direct smoking Bearing in mind the large number of variables that time, the kind of wood employed and all the raw materials in fluence the smoking process, such as the unpredictable used. 10 E. Ledesma et al .

Conclusions European Regional Development Fund (ERDF) [The Foundation for the Promotion in Asturias of Applied Scienti fic An increase in smoking time from 0 to 7 days produces Research and Technology (FICYT), grant number IE07-161, opposite effects on the moisture and the BaP content of grant number ITE09-068 (“Jovellanos Programme”)]. chorizo. While the moisture content decreases (from 49.9% ± 3.2% to 31.3% ± 1.2%), the BaP content increases –1 –1 (from less than 0.24 µg kg to 0.75 ± 0.05 µg kg ), finally References becoming stabilised after 5 days of smoking. After this [ATSDR] Agency for Toxic Substances and Disease Registry time, BaP is mainly deposited in the casing of the product. [Internet]. 1995. Toxicological pro file for polycyclic aromatic The BaP content decreases and the moisture content hydrocarbons. US Department of Health and Human Services- increases progressively from the casing Public Health Service; [cited 1995 Aug]. Available from: fi (20.0 ± 1.1 µg kg –1, 12.4% ± 1.4%) towards the inside of http://www.atsdr.cdc.gov/toxpro les/tp69.pdf –1 [ATSDR] Agency for Toxic Substances and Disease Registry. the meat product (depths D2: 1.63 ± 0.11 µg kg , 2009. Case Studies in Environmental Medicine Toxicity of –1 29.2% ± 1.5%; D3: 1.12 ± 0.10 µg kg , 35.5% ± 0.3%; Polycyclic Aromatic Hydrocarbons (PAHs) [Internet]; [cited and D4: 0.88 ± 0.10 µg kg –1, 33.8% ± 1.3%). 2009 Jul 1]. Available from: http://www.atsdr.cdc.gov/csem/ Obstruction of the pores of the casing could be caused pah/docs/pah.pdf by smoke components, making this casing a barrier pre- Andrée S, Jira W, Schwind KH, Wagner H, Schwägele F. 2010. Chemical safety of meat and meat products. Meat Sci. venting further PAHs contamination of the foodstuff. 86:38–48. However, degradation of the casing during a long smoking Basu P. 2010. Biomass Gasi fication and Pyrolysis Practical time may produce the opposite effect. design and theory. Burlington (MA): Elsevier. All the chorizo samples (without casing) studied in [CAC] Codex Alimentarius Commission. 2009. Code of practice this paper will meet the current (5 µg kg –1) and future for the reduction of contamination of food with polycyclic –1 aromatic hydrocarbons (PAH) from smoking and direct drying (2 µg kg ) BaP limits stipulated in European Union processes (CAC/RCP 68/2009) [Internet]; [cited 2009]. Commission (EU) Regulation No. 835/2011, to be applied Available from: http://www.codexalimentarius.org/standards/ from 1 September 2014. With a content of list-ofstandards/en/?provide=standards&orderField=fullReference 20.0 ± 1.1 µg kg –1, the casing of the smoked product &sort=asc&num1=CAC/RCP (D1) is four times in excess of the current legal limit. Chiu CP, Lin YS, Chen BH. 1997. Comparison of GC-MS and HPLC for overcoming matrix interferences in the analysis of PAH contamination of traditional smoked meat products PAHs in smoked food. Chromatographia. 44:497 –504. is dif ficult to control because the direct smoking process is Djinovic J, Popovic A, Jira W. 2008. Polycyclic aromatic hydro- affected by a great number of parameters such as the time of carbons (PAHs) in different types of smoked meat products smoking, the distance between the heat source and the from Serbia. Meat Sci. 80:449–456. smoked product, the amount of fat in the product and the European Union Commission (EC). 2006. Commission Regulation (EC) Nº 1881/2006 of 19 Dec 2006 setting max- kind of wood. However, a low BaP content has been found in imum levels for certain contaminants in foodstuffs (EC, No recent studies of traditional direct smoked meat products, 1881, 2006) [Internet]; [cited 2006 Dec 20]. Available from: indicating that it can be achieved (Jira et al. 2006 ; Purcaro http://eur-lex.europa.eu/LexUriServ/LexUriServ.do?uri=OJ: et al. 2009 ; Santos et al. 2011 ; Roseiro et al. 2012 ). L:2006:364:0005:0024:EN:PDF Information on innovate smoking technologies or the intro- European Union Commission (EU). 2011. Regulation Nº 835/ fi 2011 of 19 August 2011 amending Regulation (EC) No duction of simple modi cations in the smoking process will 1881/2006 as regards maximum levels for polycyclic aro-

Downloaded by [Manuel Rendueles] at 11:04 26 August 2014 be necessary in some cases to meet the forthcoming max- matic hydrocarbons in foodstuffs (EC, No 835, 2011) imum amount of BaP allowed in smoked meat products by [Internet]; [cited 2011 Aug 20]. Available from: http://eur- the European Union regulation, i.e. 2 μg kg –1, such as repla- lex.europa.eu/LexUriServ/LexUriServ.do?uri=OJ:L:2011:21 cing direct smoking methods by indirect techniques, e.g. 5:0004:0008:EN:PDF Falcó G, Domingo JL, Llobet JM, Teixidó A, Casas C, Müller L. friction smoke generation. 2003. Polycyclic aromatic hydrocarbons in foods: human exposure through the diet in Catalonia, Spain. J Food Protect. 66:2325–2331. Acknowledgements Fontcuberta M, Arques JF, Martinez M, Suarez A, Villalbi JR, Centrich F, Serrahima E, Duran J, Casas C. 2006. The authors gratefully acknowledge the support of the Centre for Polycyclic aromatic hydrocarbons in food samples col- the Development of Industrial Technology (CDTI) and the lected in Barcelona, Spain. J Food Protect. 69:2024 – Foundation for the Promotion of Scienti ﬁc and Applied 2028. Research and Technology in Asturias (FICYT). (FAO) Food and Agriculture Organization of United Nations. Agriculture and Consumer Protection Department (2013). Meat & Meat Products [Internet]; [cited 2013 Mar 1]. Funding Available from: http://www.fao.org/ag/againfo/themes/en/ This work was supported by the Spanish Ministry of Science and meat/home.html Innovation (MICINN) [The Centre for Industrial Technological Fretheim K. 1976. Carcinogenic polycyclic aromatic hydrocar- Development (CDTI) grant number IDI 20091089] and the gov- bons in Norwegian smoked meat sausages. J Agr Food ernment of the Principality of Asturias co- ﬁnanced by the Chem. 24:976–979. Food Additives & Contaminants: Part A 11

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4.3 INFLUENCIA DEL TIPO DE TRIPA EN LA PENETRACIÓN DE BENZO(A)PIRENO EN CHORIZO AHUMADO

El trabajo anteriormente presentado indica que la mayor cantidad de BaP se queda depositado en la tripa natural sin penetrar en el interior del chorizo asturiano ahumado a modo directo. Algunos estudios realizados hace algunos años proponían que el uso de distintos tipos de tripa podría influenciar la contaminación de los productos. Por ello, el estado del arte actual está enfocado en estimar la influencia del uso de distintos tipos de tripa, en el contenido de BaP y HAP en productos cárnicos embutidos.

En el trabajo expuesto a continuación se publicaron por primera vez las causas que producen las diferencias en la contaminación por BaP de chorizos ahumados embutidos con distinto tipo de tripa, natural (ChN) y sintética (ChS). Para ello se realizaron varios estudios novedosos. En primer lugar se diseñó un sistema basado en aceite embutido para eliminar las interferencias que causan las distintas profundidades, de acuerdo a los resultados encontrados en el trabajo anterior. Así mismo, se realizaron los estudios de contenido de BaP en ChN y ChS, controlando el resto de variables influyentes. Por otro lado, se diseñó un dispositivo que permite secar las tripas de manera controlada y semejante al proceso de ahumado industrial, con el objetivo de determinar la porosidad de distintos tipos de tripa mediante porosimetría. Así mismo se estudió la evolución de las tripas y ChN y ChS durante el ahumado directo, mediante el uso de microscopía electrónica de barrido (SEM) y estereomicroscopía de fluorescencia óptica. En el trabajo se encontraron y publicaron por primera vez las diferencias físicas que ocurren entre ChN y ChS durante el procedimiento de ahumado, causantes de la contaminación por HAP. Con estos resultados se detalló el mecanismo de contaminación por HAP del chorizo asturiano ahumado.

Publicación: Ledesma, E., Rendueles, M., & Díaz, M. (2015). Characterization of natural and synthetic casings and mechanism of BaP penetration in smoked meat products. Food Control, 51, 195-205.

Situación: Artículo publicado en Food Control (Elsevier).

137

Food Control 51 (2015) 195 e205

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Food Control

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Characterization of natural and synthetic casings and mechanism of BaP penetration in smoked meat products

Estefanía Ledesma a, b, Manuel Rendueles a, *, Mario Díaz a a Department of Chemical Engineering and Environmental Technology, University of Oviedo, Faculty of Chemistry, C/Juli an Clavería s/n, 33071 Oviedo, Principality of Asturias, Spain b Research, Development and Innovation (R &Dþi) Department, El H orreo Healthy Food S.L, C/Las Cabanas~ 43-45, 33180 Nore na,~ Principality of Asturias, Spain article info abstract

Article history: Meat product casings are able to prevent the penetration of chemical and potentially carcinogenic Received 6 August 2014 compounds. Some components play an important role in the food safety of smoked meat products. In Received in revised form this study, the penetration of benzo(a)pyrene (BaP) (a polycyclic aromatic hydrocarbon [PAH] marker) in 27 October 2014 smoked meat products stuffed in different casings (natural and synthetic) has been tested. It was found Accepted 15 November 2014 that, in contrast to natural casing, collagen casing prevents BaP contamination of smoked meat products. Available online 26 November 2014 The reasons of this fact have never been explained. Porosimetry and physical morphology determination by means of scanning electron microscope (SEM) and Fluorescence Stereo Microscope images of both Keywords: Benzo(a)pyrene (BaP) types of unsmoked and smoked casings and different depths of 48 chorizos stuffed with both casings e Smoked meat product subjected to 0 10 days of smoking were studied to explain this effect. Based on the results, a mechanism Type of casing can be inferred to explain BaP penetration inside the products. During smoking, meat products are Casing porosity heated and dried. The wrinkled morphology and high porosity (66.8%) of natural casing makes the fat Casing morphology ﬂows outward through the casing making its surface sticky. Soot particles and tar aerosols containing polycyclic aromatic hydrocarbons (PAH) stick to this surface and then start to migrate inwards. In contrast, the smooth morphology and low porosity (16.6%) of synthetic casing make it a barrier, impeding the fat from exiting the inside of the casing. In this case, the product shows a hard, dry external surface with less af ﬁnity for soot particles, whose possibility of penetrating the product is hindered by the small pore size of the casing. © 2014 Elsevier Ltd. All rights reserved.

1. Introduction During uncontrolled smoking, however, many chemical contaminants are formed, including polycyclic aromatic hydrocarbons The growing demand for meat, meat products and other (PAH), dioxins, formaldehyde, nitrogen and sulphur oxides (rele- protein-rich food sources in many parts of the world is of increasing vant for the formation of nitrosamines, for instance) and even concern in the light of the growing population figures, environ- heavy metals ( CAC, 2009 ). The World Health Organization (WHO), mental sustainability issues and land-use and food safety concerns. International Agency for Research on Cancer (IARC), European Food Questions related to optimal production and processing methods, Safety Authority (EFSA) and US Environmental Protection Agency location, health effects, environmental impact and legal issues (EPA) have warned of the carcinogenic activity of PAH. The most remain unanswered ( EC, 2013; FAO, 2013 ). One of the oldest pro- significant endpoint of PAH toxicity is cancer ( ATSDR, 1995; ATSDR, cesses invented by humans to extend the storage period of the 2009 ). The Commission of the European Communities (EC) has aforementioned products, maintaining their quality and providing recently amended Regulation (EC) No. 1881/2006 ( EC, 2006 ) by flavour and the consistency required by consumers is smoking. means of Regulation (EU) No. 835/2011 of 19 August 2011 ( EC, 2011 ), setting new maximum levels for PAH in foodstuffs. This new regulation speci fies that two dates must be considered as regards PAH contamination in foodstuffs. New maximum levels for * Corresponding author. Tel.: þ34 985103439; fax: þ34 985 103434. E-mail addresses: [email protected] (M. Rendueles), [email protected] the sum of four substances (PAH4) (benzo(a)pyrene, benzo(a) (M. Díaz). anthracene, benzo(b)fluoranthene and chrysene) were introduced, http://dx.doi.org/10.1016/j.foodcont.2014.11.025 0956-7135/ © 2014 Elsevier Ltd. All rights reserved. 196 E. Ledesma et al. / Food Control 51 (2015) 195 e205 maintaining a separate maximum level for BaP to ensure compa- immersion in water, as these processes may remove PAH- rability between previous and future data. The maximum permis- containing soot and particles on the surface of the food. It has sible BaP content in smoked meat and smoked meat products was been found that the greatest amount of BaP (and probably other 5.0 mg/kg in wet weight until 31/8/2014 and is 2.0 mg/kg in wet PAH) is deposited in the meat product casing and then migrates weight from 1/9/2014 on. The maximum permissible sum of PAH4 into the product ( Andree, Jira, Schwind, Wagner, & Schwagele, in these foods was 30.0 mg/kg in wet weight from 1/9/2012 to 31/8/ 2010; Djinovic et al., 2008 ; García-Falcon & Simal-Gandara, 2005 ; 2014 and is 12.0 mg/kg in wet weight from 1/9/2014 on. Although Ledesma et al., 2014; Santos et al., 2011 ). Some authors report that humans may be exposed to PAH because of environmental condi- the use of different types of casing (natural and synthetic) has an tions, the main source of contamination is diet ( Falc o et al., 2003; in fluence on the PAH content of meat products ( Filipovic & Toth, Ib anez~ et al., 2005; Lodovici, Dolara, Casalini, Ciappellano, & 1971; Toth, 1973 ). Some studies have recently continued this line Testolin, 1995; Martorell et al., 2010; Phillips, 1999; Skupinska, of research, finding different results ( Gomes, Santos, Almeida, Elias, Misiewicz, & Kasprzycka-Guttman, 2004 ). Due to their wide- & Roseiro, 2013; P ohlmann€ et al., 2013b; Skaljac et al., 2014 ), spread consumption, the major contributors to PAH intakes in diet although the reasons underlying this effect have yet to be studied. are cereals, cereal products, vegetable fats and oils. The aim of this study was to test the in fluence of the type of There has been a great deal of concern about the PAH content in casing (natural or synthetic) on BaP penetration in a typical Spanish meat products, although low values of below 1 mg/kg have recently smoked meat product called “chorizo”, determining the physical been reported ( Jira, Ziegenhals, & Speer, 2006; Purcaro, Moret, & differences between the two casings and proposing a mechanism to Conte, 2009; Roseiro, Gomes, Patarata, & Santos, 2012; Santos, explain PAH contamination of stuffed meat products during the Gomes, & Roseiro, 2011 ). Lorenzo et al. (2011) also found low BaP smoking process. levels and concluded that BaP is a good marker for the S 15 US EPA PAH in “Chorizo gallego” samples as well as for 7 US EPA probably 2. Materials and methods carcinogenic PAH in “Chorizo gallego” and “Chorizo de cebolla ” samples. PAH are mainly accumulated in the fat content of foods. 2.1. BaP determination in smoked oil, casing and meat samples Therefore, oils have been found to be one of the highest PAH- contaminated foods. This fact is mainly due to atmospheric pollu- The BaP content of meat products decreases progressively from tion contaminating the raw ingredients ( Guillen, Sopelana, & the casing to the centre of the meat product. For this reason, Palencia, 2004; Rodríguez-Acu na,~ P erez-Camino, Cert, & Moreda, samples from different products must be taken from the same 2008 ). Meat product mixtures after processing are also not product depth and location to compare results. Analysis of samples exposed to smoking processes. However, olives and meat product with or without casing must be speci fied ( Ledesma et al., 2014 ). In ingredients can become PAH contaminated because of exposure of this work 2 methods have been applied to test penetration of BaP in their skin or surface to polluted air or, in the case of olives, during different natural and synthetic casings. With the aim of create an milling as well ( Fromberg, Højgård, & Duedahl-Olesen, 2007; ideal system of BaP dilution without product depths interferences, Rodríguez-Acu na~ et al., 2008 ), resulting in these carcinogenic a system of oil stuffed in casings was developed. On the other hand, compounds appearing in the final products (CAC, 2009; Rodríguez- real meat products were analyzed to con firm results. Acuna~ et al., 2008 ). The “Code of practice for the reduction of contamination of food 2.1.1. Oil samples preparation with (PAH) from smoking and direct drying processes ” (CAC/RCP 400 mL of extra virgin olive oil (EVOO) (Manufactured by Car- 68/2009) stipulates that PAH contamination in foodstuffs via food bonell, Spain) were poured into 40 cm of each one of 2 types of processing should be minimized, providing producers with the casings, natural (from pig intestine) and synthetic (collagen) pre- variables that need to be controlled. These variables have been viously wetted in salty water. 40 mL of unprocessed oil were kept in studied by several authors, con firming that the PAH content of meat the original bottle, in the dark, for analysis. The samples were hung products can be reduced by means of its control. They include the 40 cm apart on a bar, placed in the same position and location in the kind of fuel ( Hitzel, Pohlmann,€ Schw agele,€ Speer, & Jira, 2013; smoking chamber, at the same distance from the smoke source, Pohlmann,€ Hitzel, Schw agele,€ Speer, & Jira, 2012; Stumpe-V ıksna, namely 10 m, and smoked for 7 days employing a mixture of oak Bartkevics, Kukare, & Morozovs, 2008 ), smoking method (90%) and chestnut (10%). Once smoked, samples were hung on a (Pohlmann€ et al., 2012, P ohlmann,€ Hitzel, Schw agele,€ Speer, & Jira, bar placed into a box and sent for laboratory analysis. The oil 2013a; Skaljac et al., 2014; Varlet et al., 2007 ), smoking duration content of the samples and the casings were subsequently kept (Djinovic, Popovic, & Jira, 2008; Essumang, Dodoo, & Adjei, 2013; separately in individual glass flasks in a fridge, at 5 C in the dark. Ledesma, Rendueles, & Díaz, 2014 ), fat content ( Pohlmann,€ Hitzel, Fifteen samples were finally analyzed, three for each one of the Schwagele,€ Speer, & Jira, 2013b ), distance between the food and following products: original oil, smoked oil stuffed in natural cas- the heat source, position of the food in relation to the heat source ing, smoked oil stuffed in synthetic casing, smoked natural casing, (Pohlmann€ et al., 2013b ), temperature during smoking and direct and smoked synthetic casing. drying (Pohlmann€ et al., 2012 ), cleanliness and maintenance of equipment and the design of the smoking chamber, and the 2.1.2. Meat products preparation equipment used for the smoke/air mixture ( Pohlmann€ et al., Chorizo is a typical Spanish smoked meat product made from 2013a ). The use of woods from apple ( Stumpe-V ıksna et al., the following ingredients: pork meat, pork fat, salt, garlic, sweet or 2008 ), oak and hickory ( Hitzel et al., 2013 ), indirect smoking spicy paprika and herbs. Antioxidants such as sodium citrate or methods, like friction smoke generation (Pohlmann€ et al., 2013a ), sodium ascorbate, colourings such as carmine, emulsi fiers such as reducing smoking time (Djinovic et al., 2008; Ledesma et al., 2014 ), sodium triphosphate, food preservatives such as sodium nitrite and reducing fat content (Pohlmann€ et al., 2013b ), reducing smoke other food additives were used to ensure the microbiological sta- generation temperatures (below 600 C) ( Pohlmann€ et al., 2012 ), or bility and quality of the foodstuff. Two kinds of chorizo were pre- put the products in front, centre-front position of smoking chamber pared for this study: chorizo encased in natural casing, and chorizo (Pohlmann€ et al., 2013b ) helps to reduce final BaP and PAH contents encased in synthetic casing. Fifty kg of each chorizo were manu- of meat products. Moreover, as a post-smoking treatment, CAC factured at the El H orreo Healthy Food S.L. meat company facilities (2009) advises cleaning the product by means of rinsing or in the following steps: reception of raw ingredients, mincing, E. Ledesma et al. / Food Control 51 (2015) 195 e205 197 mixing, marinating, stuffing, direct smoking, packaging and label- et al., 2009; Stumpe-V ıksna et al., 2008 ). Finally, the samples ling. The chorizo samples were stuffed in strings of 4 chorizos. A were sonicated for 1 h in an ultrasound bath (Cod. 3000513, J.P. mixture of oak (90%) and chestnut (10%) wood was used to smoke Selecta, S.A, Barcelona, Spain) at ambient temperature. directly the chorizo for 7 days. All the selected samples were placed in the same position in the smoking chamber, at the same distance 2.1.4.4. Filtration. After sonication, the samples were filtered on a from the smoke source, namely 10 m. Once smoked, the chorizos paper filter (reference number 1242, 11 cm, Albet) to remove the were hung in a cardboard tube and placed in a box to prevent the solid meat waste, which was washed with additional n-hexane materials from coming into contact with any other object. The (5 þ 5 mL). The solvent was subsequently collected in a flask and nutritional details of a chorizo manufactured by the company with the volume was made up to 10 mL with the help of a rotatory the same ingredients and manufacturing process as the one studied evaporator (Heidolph Laborota 4000 ef ficient, Germany) and the here, determined by the Principality of Asturias Meat Industry addition of the necessary amount of n-hexane. Association Technological Centre for Supporting Innovation (Spanish acronym, ASINCAR), are 31.41% moisture content, 22.98% 2.1.4.5. Solid phase extraction (SPE) step. The extract obtained after protein, 37.94% fat, 2.85% carbohydrates, 4.82% ash and pH 5.2. Six sonication contained an appreciable amount of fat ( Purcaro et al., samples were finally analyzed, three for each of the following 2009 ). A solid phase extraction (SPE) procedure in a Supelco Visi- products: smoked chorizo stuffed in natural casing, and smoked prep TM SPE vacuum manifold with 12 ports (Sigma eAldrich, USA), chorizo stuffed in synthetic casing. was used to isolate benzo(a)pyrene and benzo[a]pyrene-d12 (BaP- d12) (the sought-after PAH in the sample), similar to the previously 2.1.3. Reagents and standards established setup for rapid PAH determination in vegetable oils by Benzo[a]pyrene (BaP) analytical standard solution was supplied Moret and Conte (2002) . First, a 5 g silica SPE cartridge (Mega Bond by Sigma eAldrich at a concentration of 100 mg/mL in cyclohexane Elut, 20 mL, Varian) was slowly washed with 20 mL of dichloro- and a total volume of 2 mL. Benzo[a]pyrene internal standard (d12, methane, dried completely under vacuum and conditioned with 98%) solution was obtained from Cambridge Isotope Laboratories at 20 mL of n-hexane (to prevent the cartridge from drying). Next, a concentration of 200 mg/mL in isooctane and a total volume of 1 mL of the sonicated sample was diluted in 3 mL of n-hexane and 1.2 mL. All solvents for sample preparation, hexane, dichloro- slowly loaded into the cartridge, avoiding complete drying of the methane, cyclohexane and isooctane (2, 2, 4-Trimethylpentane), cartridge. The aliphatic hydrocarbons were then discharged via the were purchased from Sigma eAldrich with ACS reagent addition of 8 mL of a mixture of hexane and dichloromethane 70:30 purities ! 99% (GC) grade. Solid phase extraction (SPE) cartridges (v/v) to the cartridge. Finally, after loading the cartridge with were supplied by Varian and Agilent Technologies. The Mega Bond another 8 mL of the hexane and dichloromethane 70:30 (v/v) so- Elut cartridges employed contain a silica sorbent phase, with a lution, the PAH, BaP and BaP-d12 contained in the sample were particle size of 40 nm, a weight of 5 g and a volume of 20 mL. slowly discharged into 15 mL amber glass vials, allowing the car- Amber-coloured flasks and vials were used to protect the PAH from tridge to dry under vacuum. light decomposition. Specifically, 15 mL (21 Â 70 mm) amber glass vials were purchased from Sigma eAldrich for PAH collection dur- 2.1.4.6. Concentration step. The samples were concentrated after ing the solid phase extraction (SPE) step. the SPE step, the volume being made up to 1 mL by N 2 blowing using a nitrogen stream at the top of 15 mL volume vials placed in a 2.1.4. Sample pre-treatment of chorizo samples fume hood. Finally, the samples were loaded into 2 mL GC/MS 2.1.4.1. Grinding step. First, the chorizo casing was removed and amber glass vials and the final volume of 1 mL was checked by stored for its analysis, following the procedures speci fied by weighing the vials. Fretheim (1976) and Wretling, Eriksson, Eskhult, and Larsson (2010) . Then, samples were cut in slices and the amount of cho- 2.1.4.7. Sample pre-treatment of oil samples. A solid phase extrac- rizo situated at a distance between 0.25 cm from the casing and the tion (SPE) procedure was used to isolate benzo(a)pyrene and benzo casing of the chorizo, denominated depth 2 (D2) according to [a]pyrene-d12 (BaP-d12), similar to that previously described for Ledesma et al. (2014) was taken. 20 g of D2 were finely homoge- meat product pre-treatment. 2.5 g ± 0.001 g of oil were weighed nised in a meat grinder (Minirobot D81 meat grinder, Moulinex, into a 10 mL volumetric flask, 200 mL of benzo[a]pyrene-d 12 inter- France) following the protocol developed by Purcaro et al. (2009) . nal standard solution at a concentration of 100 mg/L were then Pre-treatment of all samples was carried out in triplicate. added and the volume was made up to 10 mL with n-hexane. This solution was carefully stored under refrigeration for 3 days to help 2.1.4.2. Lyophilisation step. Three aliquots of 20 g chorizo were the deuterated compound adapt to the sample matrix. Then, 1 mL taken, placed in individual lyophilisation plates carefully sealed of the sonicated sample was slowly loaded into the cartridge and with aluminium foil and weighed before freeze-drying. Each sam- the same SPE and concentration procedures, as described for meat ple was then frozen to 193.15 K for 4 h and freeze-dried in a product pre-treatment, were applied. lyophilizer (Telstar-Cryodos, Spain) for 1 day. Finally, the plates (containing the sample) were weighed after freeze-drying. 2.1.4.8. Sample pre-treatment of casing samples. Casings were placed in flasks, adding 20 mL of n-hexane and 200 mL of benzo[a] 2.1.4.3. Sonication step. Two grams of freeze-dried chorizo (one pyrene-d 12 internal standard solution at a concentration of 100 mg/ from each lyophilisation plate) were placed in flasks (the remaining L. This solution was carefully stored under refrigeration for 3 days. amount of lyophilised chorizo being stored in a freezer). Subse- The samples were then treated following the same procedure as quently, 20 mL of n-hexane and 200 mL of benzo[a]pyrene-d 12 in- chorizo samples from the sonication step on. ternal standard solution at a concentration of 100 mg/L were added (dilutions from the stock solution must be made previously). This 2.1.5. BaP determination by GS/MS solution was carefully stored under refrigeration for 3 days to help 2.1.5.1. GC/MS calibration. BaP quanti fication was performed via the deuterated compound adapt to the sample matrix. This is the prior calibration of the GC/MS system with benzo(a)pyrene and appropriate moment to add the internal standard compound as it is benzo[a]pyrene-d12 standard solutions. For calibration purposes, the first time both compounds are in the same matrix ( Purcaro benzo[a]pyrene-d12 standard solution was added to check 198 E. Ledesma et al. / Food Control 51 (2015) 195 e205 extraction recovery during the pre-treatment methods. Calibration casing, natural (from pig intestine) and synthetic (collagen), were curves were generated by injecting 6 diluted standard solutions of wetted in the same salty water. Meanwhile, the central section of a BaP and BaP-d12 in cyclohexane, in triplicate. BaP standard con- 53 cm hollow cardboard tube was removed to create a space with centrations for each calibration vial were 0.05 ppb, 0.1 ppb, 0.5 ppb, no material where air could pass through after making a hole in the 1.0 ppb, 5 ppb and 10 ppb. The concentration of BaP-d12 for all tube. Then the surfaces of the cardboard tube were covered with a calibration vials was 2 ppb. The MS assistant program (Agilent plastic parafilm to ensure the casings did not slip, thus preventing Technologies) compares the relationship between both BaP stan- their degradation. Then, systems of casings were hung on inde- dard signals (BaP and BaP-d12) to determine the final BaP con- pendent bars and placed on a metal shelve and slowly and carefully centration. Identification of compound was assessed comparing the dried for 3 days in a controlled-atmosphere drying shed, with retention time and confirm the ion ratios (quali fier ion/target ions) temperature and humidity conditions of 285.15 K and 65%, with a criterion of ±20% of standards compounds ratio. Criterion of respectively. Fig. 1 shows the system. Once dried, the samples were BaP/BaP-d12 standards target ion ratios was ±20%.A blank (i.e. a vial carefully transported hung on bars placed inside a box, preventing containing only the solvent cyclohexane, with no BaP or BaP-d12 their contact with any other object. standard compounds) was also included among all the standard vials containing BaP compounds. 2.2.2. Determination of casing porosimetry 0.51 g and 0.42 g of dried synthetic and natural casing, respec- 2.1.5.2. Gas chromatography (GC). Separation of BaP and BaP-d12 tively, were analyzed by mercury intrusion porosimetry using a was performed on an HP-5MS 19091S-433 part number column Micromeritics Autopore IV device. Analyses were performed under (30 m Â 250 mm Â 0.25 mm) (Agilent Technologies) in an Agilent pressure values ranging from 0.10 to 60,000.00 psia on previously Technologies 6890 gas chromatograph (GC). The injection tem- evacuated samples to obtain the following material characteristics: perature and volume were 300 C and 1 mL (splitless), respectively. all the intrusion data, pore structure data and material compress- Helium was applied as carrier gas at a constant flow rate of 1.5 mL/ ibility data. min. The temperature ramp used was the following: isothermal at 55 C for 1 min, increasing at a rate of 25 C/min to 320 C for 3 min, 2.3. Microscopic morphology of casings and meat products total run time 14.60 min. A Jeol 6100 scanning electron microscope operating at 30 kV fi 2.1.5.3. Mass spectrometry (MS). Identi cation of BaP and BaP-d12 was used to determine the microscopic morphology of the was performed using an Agilent Technologies 5975 mass spec- following samples: both sides of the casings pre-treated as stated in trometer detector (MS) equipped with a G2589-20045 part number Section 2.2; and the natural and synthetic casings of eight chorizos 6-mm ultra-large aperture drawout lens. The solvent delay was smoked after 3 and 5 days, manufactured as stated in Section 2.1.2 4 min and the EM voltage (run at autotune voltage) was 1294 V. The and pre-treated by freezing to 193.15 K for 4 h and freeze-drying in low mass was 252 amu and the high mass was 264 amu. SIM mode a lyophilizer (Telstar-Cryodos, Spain) for 1 day. was selected for 1 group, 3 ions per group (252, 264 and 126), 50 A Leica M205 FA Fluorescence Stereo Microscope was used to msec dwell/ion. The quad temperature was 180 C, the source determine the microscopic morphology of the following samples: temperature, 300 C and the transfer line temperature, 280 C. Data the natural and synthetic casings pre-treated as stated in Section were acquired by operating the MS in ion monitoring mode. Peak 2.2; the natural and synthetic casings of 8 unsmoked chorizos (4 for spectra were compared to the mass spectra of BaP and BaP-d12 each type of casing); and 40 chorizos smoked between 0 and 10 standards and the library supplied with the instrument. Ten days (20 for each type of casing) and their external and internal different spiked samples (as well as unspiked controls), were pre- surfaces (once the casing was removed), manufactured as stated in pared and each of these samples and vials was analyzed in triplicate Section 2.1.2 . according to the analytical procedure. Recoveries were calculated by comparing the difference between spiked and unspiked samples 2.4. Statistical analysis with the known amount of added BaP standard (1 ppb). Detection fi and quanti cation limits (LODs and LOQs, respectively) were Data from each study were analysed by running Fisher's least evaluated on the basis of the noise obtained through analysis of the significant difference (LSD) procedure for multisample statistical blank samples. LOD and LOQ were de fined as the concentration of the analyte that produced a signal-to-noise ratio of 3 and 10, respectively. Moreover, these results were con firmed by the analysis of blank samples and 6 diluted standard solutions of BaP and BaP-d12 in cyclohexane, in triplicate. The slope ( m) and standard deviation of intercept (sb 0) of the analytical response ( y) vs concentration level added ( x) were calculated, then the LOD and LOQ were also estimated via the following equations: LOD ¼ 3.3*sb0/m, LOQ ¼ 10*sb 0/m.

2.2. Porosimetry determination of natural and synthetic casings

2.2.1. Casing samples preparation Synthetic casings are marketed in the dry state, while natural casings have a high moisture content. Porosimetry equipment does not allow wet samples analysis. The lyophilisation method was dismissed because of possible degradation and transformation of the samples. For this reason, a special system was prepared to dry casings in the same way as meat products, avoiding contamination of the casings by ingredients. Sixty centimetres of each type of Fig. 1. Casings slow drying system. E. Ledesma et al. / Food Control 51 (2015) 195 e205 199 comparison. This method was used to discriminate between the Portuguese market. A content of 0.08, 0.18 and 0.04 mg/kg of BaP means. Furthermore, t-tests between consecutive samples were was found by Djinovic et al. (2008) in unsmoked Sremska and Cajna applied to compare means, at signi ficant level (p < 0.05). F-tests at sausages and pork ham, respectively. After 5 days of exposure to significant level ( p < 0.05) were previously carried out to compare smoking, different BaP contents were found in oils and meat standard deviations. Standardized skewness and standardized products stuffed in natural and synthetic casings. While an amount kurtosis were used to assess whether the samples came from of 2.16 ± 0.12 mg/kg was found in the oil stuffed in the natural normal distributions. The software used was STATGRAPHICS Plus casing, BaP was not found in the smoked oil stuffed in the synthetic 3.1. casing. However, a great amount of BaP was found in the external part of both the natural (19.9 ± 2.9 mg/kg) and synthetic 3. Results and discussion (14 .0 ± 0.2 mg/kg) casing. Significant differences (p < 0.05) between BaP content in both casings were found. 3.1. BaP content of oil and chorizo stuffed in natural and synthetic Likewise, BaP was not found in smoked meat products stuffed in casings collagen casings, although 1.71 ± 0.15 mg/kg was found in the D2 depth of products stuffed in natural casings. All the samples were successfully analysed by the proposed Significant differences ( p < 0.05) were found in the BaP content methods. Previous studies consisting of the combination of soxhlet of oils and chorizos stuffed in natural casings. and Gel Permeation Chromatography (GPC) were performed in Similar results of BaP content in natural casing (20.0 ± 1.14 mg/ order to evaluate analytical methods for BaP determination in kg) and D2 depth of chorizo (1.63 ± 0.11 mg/kg) where found in smoked meat products. The combination of sonication and SPE pre- previous works ( Ledesma et al., 2014 ). treatment methods to determine the BaP content in smoked meat These results are in line with recent studies, the main amount of products by GC/MS analysis was found to be a good method, as it PAH remains in the casing and does not penetrate inside the meat entails a low level of solvent consumption and waste generation, product ( Andree et al., 2010; Djinovic et al., 2008 ; García-Falcon & short operating times and is easy to set up. A similar retention time Simal-Gandara, 2005 ; Gomes et al., 2013; P ohlmann€ et al., 2013b; was found for the BaP and BaP-d12 standards, respectively 12.47 s Santos et al., 2011 ), and then higher level of PAH was found in- and 12.49 s. Ion ratios in all samples were within the required side the meat products stuffed in natural than in collagen casings criterion (±20) and compounds were con firmed. This method offers smoked under the same conditions (Gomes et al., 2013; Lorenzo high quality quantitative results with high extraction ef ficiency. et al., 2011; Roseiro et al., 2012; Santos et al., 2011; Skaljac et al., The limit of detection (LOD) for both compounds was 0.05 mg/kg 2014 ). However, a recent study by Pohlmann€ et al. (2013b) re- and the limit of quanti fication (LOQ) was 0.24 mg/kg. Recoveries ports that smoked Frankfurter sausages with natural (sheep in- ranged from 98% to 101% and relative standard deviations (RSDs) testine) casing had a similar BaP and PAH4 content to those with a were lower than 4%. collagen casing while lower levels of these contaminants were The BaP content found in the unsmoked oil system and meat found in Frankfurter sausageswith peelable cellulose casings. The products, smoked meat products and oils stuffed into natural and amount of BaP found in the tested chorizos falls well below the synthetic casings are shown in Table 1 The oil system was prepared to test the penetration of BaP across natural and collagen casings. As Table 1 shows, BaP was not found in the unsmoked oil or in the Table 2 oil stuffed in the collagen-casing system, while it did appear in the Intrusion data, pore structure and Mayer Stowe summaries of natural (from pig system prepared with natural casing. The same effect was also intestine) and synthetic (collagen) casings. found in meat products. The absence of BaP in all unsmoked Casing samples is a coherent result, as EVOO is not exposed to PAH during its production processes. However, PAH can be found in unsmoked Natural Collagen oil and also in raw sausage mixtures due to environmental Intrusion data summary À1 contamination of ingredients ( CAC, 2009; Djinovic et al., 2008; Total intrusion volume (mL g ) 1.37 0.14 Total pore area (m 2 gÀ1) 9.11 11.5 Rodríguez-Acu na~ et al., 2008 ). According to reported results, a Median pore diameter (Volume) (nm) 66018.7 44815.1 range level between less than 0.2 mg/kg and 0.4 mg/kg of BaP was Median pore diameter (Area) (nm) 4.90 5.10 found by Fromberg et al. (2007) in 46 samples of merchant EVOO Average pore diameter (4 V/A) (nm) 599.8 48.2 for human consumption from Italy, Spain, Greece, France and Bulk density at 1.49 psia (g mL À1) 0.49 1.20 À1 Holland. A content of between 0.07 and 0.28 mg/kg of BaP was found Apparent (skeletal) density (g mL ) 1.47 1.44 Porosity (%) 66.8 16.6 by Teixeira, Casal, and Oliveira (2007) in 2 virgin olive oils from the Stem volume used (%) 52.0 6.00 Pore structure summary Threshold pressure (psia) 1.98 2.65 Table 1 Characteristic length (nm) 91261.6 68141.3 BaP content (mg/kg) of the studied samples: unsmoked extra virgin olive oil, smoked Conductivity formation factor 0.36 0.07 extra virgin olive oil, and meat products stuffed into natural (from pig intestine) and Permeability constant 0.00442 0.00442 synthetic (collagen) casings ( n ¼ 3). Permeability (mdarcy) 13459.7 1380.2 BET surface area (m 2 gÀ1) 133.8 133.8 Stuffed sample BaP content (mg/kg) of Sample stuffed into this casing Pore shape exponent 1 1 Natural casing Collagen casing Tortuosity factor 1.47 2.04 Tortuosity 3.65 2.86 Unsmoked oil n.d n.d Percolation fractal dimension 3.00 2.99 Unsmoked meat n.d n.d Backbone fractal dimension 2.98 2.99 smoked oil 2.16 ± 0.12 n.d Mayer stowe summary Meat product 1.71 ± 0.15 n.d Interstitial porosity (%) 47.6 25.9 Casing only 19.9 ± 2.9* 14.0 ± 0.2* Breakthrough pressure ratio 3.35 8.32 N ¼ 3. Material compressibility n.d: non detectable. Linear coefficient (psiaÀ1) À6.21E-01 N/A *: statistical signi ficance: (<0.05). Quadratic coefficient (psiaÀ2) 4.40E-02 N/A 200 E. Ledesma et al. / Food Control 51 (2015) 195 e205 current European legal limit (2 ppb). These results are in line with collagen casing (25.9%). However, the total pore area of both casings the low BaP content found in smoked meat products reported by is similar. other authors ( Jira et al., 2006; Purcaro et al., 2009; Roseiro et al., As Table 2 shows, the total intrusion volume of the natural 2012; Santos et al., 2011 ). casing (1.37 mL/g) is higher than that of the collagen casing (0.14 mL/g). To sum up, with a higher pore diameter and total 3.2. Physical characterization of casings and meat products intrusion volume, the natural casing is four times more porous than the collagen casing. However, the tortuosity of the natural casing 3.2.1. Mercury intrusion porosimetry (3.65) is higher than that of the collagen casing (2.86). The collagen Table 2 shows the intrusion data, pore structure and Mayer- casing is made from a synthetic material, more homogeneous than Stowe data of natural (from pig intestine) and synthetic the natural casing. Most likely, a greater variation in pore diameter (collagen) casings. can be found in natural casing, making its tortuosity higher than As can be seen in Table 2 , higher values of intrusion data pa- that of the collagen material. rameters, i.e. total intrusion volume, median pore diameter (expressed in volume and area), average pore diameter, apparent 3.2.2. Scanning electron microscopy (SEM) density and finally porosity, were found in the natural casing than Fig. 2 shows SEM images of both the studied sides of natural and in the collagen casing. synthetic (collagen) casings. Fig. 2aed shows SEM images of the In fact, with a porosity of 66.8%, the natural casing is four times natural casing. Fig. 2a and b were taken on the “A” external side of more porous than the collagen casing (16.6%). The average pore the natural casing. Fig. 2c and d were taken of the “B” internal side diameter of the natural casing (599.8 nm) is considerably higher of the natural casing, that contacts with stuffed chorizo mass. than that of the collagen casing (48.2 nm). Moreover, the interstitial Fig. 2e and f shows SEM images of the synthetic casing. No differ- porosity of the natural casing (47.6%) is higher than that of the ences were found in the SEM images of both sides of the synthetic

Fig. 2. SEM images of faces “A” (a,b) and “B” (c,d) of natural casing (from pig intestine) and synthetic (collagen) casing (e,f). Scale of figures a, c and e is 500 mm. Scale of figures b, d and f is 50 mm. E. Ledesma et al. / Food Control 51 (2015) 195 e205 201 casing. As Fig. 2a and e shows, a higher number of small, elongated, magni fication) show the general morphology and appearance of white objects were found in the natural casings than in the collagen natural and synthetic smoked casings of a chorizo. Comparison of casings. These objects were identi fied as salt crystals by elemental these images indicates that the natural casing is covered with analysis by SEM (42.7% Na and 57.2% Cl). A different morphology more bodies or materials than the synthetic casing. The natural was found on the different sides of the natural casing. Side A ( Fig. 2a casing is dirtied by these materials, whereas the synthetic casing and b) shows more salt crystals than side B ( Fig. 2c and d). As Fig. 2b seems to be cleaner. Fig. 3 b and d ( Â430 magni fication) show the and d shows, side A of the natural casing is coarser and more detailed morphology and appearance of natural and synthetic wrinkled than side B. The same smooth surface was found on both smoked casings of a chorizo. The body shown on the left side of sides of the collagen casing, where the collagen fibres can be clearly the images is a soot particle. Comparison of these images indicates identified ( Fig. 2e and f). that the soot particle is embedded in the wrinkled surface of the To sum up, the natural casing has 2 sides with different surfaces, natural casing, while the collagen casing presents a smooth one of them (external side, called side A) has a wrinkled surface surface. with the capacity to capture small objects. The other (internal side, Fig. 3e and f ( Â120 magni fication) show the detailed called side B) has a smooth surface that does not capture small morphology and appearance of a soot particle from smoke found in objects. The morphology of the surface of both sides of the collagen a natural and synthetic casing of smoked chorizos, respectively. casing is similar, being flat and smooth, the arti ficial material of Comparison of these images indicates that the soot particle harms which is identi fied by collagen fibres. the natural casing, managing to penetrate into the product. In Fig. 3 shows SEM images of the natural and synthetic collagen contrast, the soot particle is deposited on the synthetic casing and casings of chorizos after 5 days of smoking. Fig. 3 a and c ( Â23 do not harm or penetrate it.

Fig. 3. SEM images of natural (from pig intestine) (a,b,e) and synthetic (collagen) (c,d,f) casings of smoked chorizos. Scale of ﬁgures “a” and “c” is 1 mm, “b” and “d” 50 mm, “e” and “f” 100 mm. 202 E. Ledesma et al. / Food Control 51 (2015) 195 e205

3.2.3. Fluorescence Stereo Microscope spheres on the surfaces. As Fig. 4d shows, the raw material remains Fig. 4 shows Fluorescence Stereo Microscope images of natural inside the collagen casing, the morphology of which does not (4a) and synthetic (4b) casings, uncured chorizo mass stuffed in present any modi fication caused by the process. The collagen casing natural (4c) and synthetic (4d) casings, chorizo smoked for 10 days is a barrier between the external and internal part of its content. stuffed in natural (4e, 4f) and synthetic (4g, 4h) casings and the Fig. 4eeh shows that, after 10 days of smoking, the surface of external (4i, 4k) and inner (4j, 4l) parts of chorizo smoked for 10 chorizos manufactured with natural and synthetic casings are very days stuffed in natural (4i, 4j) and synthetic (4k, 4l) casings, different. Fig. 4e shows that the surface of chorizos stuffed in nat- respectively. All 48 chorizos studied in this work showed a similar ural casing is full of fat that has come from inside the product, behaviour. A surface covered with a gelatinous material is found on making it sticky. Hands are soiled with fat when touching the the natural casings ( Fig. 4a), while synthetic fibres are clearly product. As Fig. 4f shows, numerous soot particles are found identified in the collagen casings ( Fig. 4b). According to Feiner embedded in the fat, which flows from inside the chorizo and (2006) natural casing is composed by several layers, organized in covers its external surface. Numerous soot particles were found in 5 sections: serosa (tunica serosa), 2 muscular layers, (tunica mus- the external and even the internal parts of chorizo when the casing cularis: stratum longitudinale and stratum circulare), submucosa was removed. The products seem to swell up. However, as can be (tela submucosa) and mucosa (mucous coat). Once the raw in- seen in Fig. 4g, the surface of synthetic chorizos is dry, does not soil gredients of the meat product are stuffed in the casings, the the hands when touched and is clean and practically free of soot collagen and natural casings are seen to behave differently. As particles, as in the internal parts of the products once the casings Fig. 4c shows, the morphologies and surfaces of the natural casings were removed. Fig. 4h shows that all the products seem to have were modi fied by their content. Once stuffed, the meat product shrunk, presenting a great deal of wrinkles on their surface. The fat mixture seems to cross the natural casings, describing small from the products remains inside. When the casings are removed,

Fig. 4. Fluorescence Stereo Microscope images of natural (a) and synthetic (b) casings, uncured chorizo mass stuffed in natural(c) and synthetic (d) casings, 10 days smoked chorizo stuffed in natural (e, f) and synthetic (g, h) casings and external (i, k) and deepest (j, l) sides of 10 days smoked chorizo stuffed in natural (i, j) and synthetic (k, l) casings respectively. The scale of all images is 500 mm, except image i, whose scale is 200 mm. E. Ledesma et al. / Food Control 51 (2015) 195 e205 203

Fig. 4. (continued).

the morphology and surface of chorizos stuffed in natural and aerosols, finally being deposited on the meat products. Meanwhile, synthetic casings are also different. As Fig. 4i shows, the surface of different processes occur in meat products stuffed in natural or natural casing of stuffed chorizos is moist, full of juicy fat, whereas synthetic casings. Both products are heated and dried during Fig. 4k shows that the fat of chorizos whose synthetic casing had smoking (Mohler,€ 1978; Varlet et al., 2007 ). However, the porosity been removed has become dried, making it hard to the touch. Fig. 4j of natural casings (66.8%) is higher than that of synthetic casings and l shows that more soot particles were found in the chorizos (16.6%). Accordingly, different effects occur in chorizos stuffed in whose natural casing had been removed than in those stripped of natural and synthetic casings. First, when stuf fing, both products their synthetic casing, where not many of these particles were swell and the raw mix of meat products passes through the natural found. Fig. 4j and l shows that, while only a few, some soot particles casing, but not through the synthetic casing. These meat products were found in the centre of the chorizos (the deepest part of a then enter the smoking chamber, where they are heated and placed section) stuffed in natural ( Fig. 4j) casings, while none was found in in contact with smoke. Heat makes chorizos stuffed in natural the innermost part of chorizos stuffed in synthetic casings ( Fig. 4l). casing sweat fat. The fat content of these chorizos flows through the casing and covers its external surface, making it sticky and moist. 3.3. Overall comments Then, when PAH aerosols reach the sticky, wrinkled surface of the natural casing of chorizos, soot particles are easily captured and All the results presented in this study help us to understand the adhere to it, damage the casing and start to migrate inside the process of BaP contamination of smoked meat products stuffed in product. Once captured, the soot particles slowly penetrate inside different casings. During traditional smoking, biomass pyrolysis the meat products, accessing the inner part through the lumps of and incomplete combustion processes occur forming undesired tar fat. Although the main amount of BaP stays in the casing, some aerosols. When the temperature rises above 1023 K, tertiary tar cross the membrane and penetrate inside the product due to the products begin to appear with increasing temperature and PAH are high porosity of the natural casing. It can be also seen that, during formed (Basu, 2010 , chap. 4). PAH are transported in these smoke the first 5 days of smoking, the membranes of the casing are more 204 E. Ledesma et al. / Food Control 51 (2015) 195 e205 moist and sticky, the product being more swollen. Hence, a greater 2009 July 1. Retrieved May 11, 2014 from http://www.atsdr.cdc.gov/csem/pah/ amount of BaP may be captured by the casing. In previous works it docs/pah.pdf. Basu, P. (2010). Biomass gasification and pyrolysis. Practical design and theory . Bur- was found that BaP increases during smoking time, finally lington, MA, USA: Elsevier. becoming stabilized after 5 days of smoking ( Ledesma et al., 2014 ). CAC (Codex Alimentarius Commission). (2009). Code of practice for the reduction of In contrast, the fat content of chorizos stuffed in synthetic casings contamination of food with polycyclic aromatic hydrocarbons (PAH) from smoking and direct drying processes (CAC/RCP 68/2009) (Cited 2009). Retrieved May 11, remains inside the product, its surface remaining dry, making it 2014 from http://www.codexalimentarius.org/standards/list-of standards/en/? non-sticky and smooth without any af finity for soot particles. provide¼standards&orderField¼fullReference&sort¼asc&num1¼CAC/RCP . Accordingly, a lesser amount of soot particles containing PAH Djinovic, J., Popovic, A., & Jira, W. (2008). Polycyclic aromatic hydrocarbons (PAH) in different types of smoked meat products from Serbia. Meat Science, 80 , (aerosols) stays on the outside of the casing and does not damage 449 e456. its surface. Moreover, the smaller size of the synthetic casing pores EC. (2006). European Union Commission (EC) (Internet). Regulation N 1881/2006 of hinders the small amount of coal tars from penetrating the product. 19 December 2006 setting maximum levels for certain contaminants in foodstuffs (EC, No 1881, 2006) (Cited 2006 December 20]. Avaiable from http://eur-lex.euro For all these reasons, high BaP levels were found on the external pa.eu/LexUriServ/LexUriServ.do?uri ¼OJ: L:2006:364:0005:0024:EN: PDF . surface of the smoked meat products stuffed in natural casing EC. (2011). European Union Commission (EC) (Internet). Regulation N 835/2011 of 19 (19.9 ± 2.9 mg/kg) and were then also found inside the meat August 2011 amending Regulation (EC) No 1881/2006 as regards maximum levels products (D2) stuffed in natural casing (1.71 ± 0.15 mg/kg). However, for polycyclic aromatic hydrocarbons in foodstuffs (EC, No 835, 2011) (Cited 2011 August 20). Avaiable from http://eur-lex.europa.eu/LexUriServ/LexUriServ.do? no BaP content was found inside (D2) the meat products stuffed in uri¼OJ: L:2011:215:0004:0008:EN: PDF . synthetic casings. These results are in keeping with the findings of EC. (2013). European Commission decision horizon 2020 work programme 2014 e Gomes et al. (2013) and Skaljac et al. (2014) , in which the use of 2015. Part 9. Food security, sustainable agriculture and forestry, marine and maritime and inland water research and the bioeconomy. Safe food and healthy synthetic instead of natural casings contributed to reducing PAH diets and sustainable consumption. SFS-15-2014: Proteins of the future (pp. levels in smoked meat products.They likewise explain the results 26 e27) (Cited 2013 December 10). Retrieved May 11, 2014 from: http://ec.euro reported by Pohlmann€ et al. (2013b) , who found only a small in- pa.eu/research/participants/data/ref/h2020/wp/2014_2015/main/h2020- wp1415-food_en.pdf . crease in BaP content in the smoked meat products stuffed in Essumang, D. K., Dodoo, D. K., & Adjei, J. K. (2013). Effect of smoke generation natural casings (0.57 ± 0.21 mg/kg) compared to collagen casings sources and smoke curing duration on the levels of polycyclic aromatic hy- (0.40 ± 0.12 mg/kg), which may be caused by prior environmental drocarbon (PAH) in different suites of fish. Food and Chemical Toxicology, 58 , 86 e94 . PAH contamination of the raw ingredients. Falc o, G., Domingo, J. L., Llobet, J. M., Teixid o, A., Casas, C., & Müller, L. (2003). Polycyclic aromatic hydrocarbons in foods: human exposure through the diet in Catalonia, Spain. Journal of Food Protection, 66 , 2325 e2331. 4. Conclusions Feiner, G. (2006). Practical science and technology: Meat products handbook (1st ed.). Cambridge, England: Woodhead publishing limited . € Natural and synthetic casings have physical differences that lead Filipovic, J., & Toth, L. (1971). Polycyclische Kohlenwasserstoffe in ger aucherten jugoslawischen Fleischwaren. “Polycyclische hydrocarbons in smoked Yugo- to different BaP content into smoked meat products. The natural slavian meat products. Fleischwirtschaft ( “Meat Economy”), 51 , 1323 e1325 casings have a wrinkled morphology and high porosity (66.8%). This (original title in German) . makes the fat content flows outward through the casing, making its Food and Agriculture Organization of United Nations (FAO). Agriculture and Con- sumer Protection Department. (2013). Meat & meat products (Cited 2013 March surface sticky and product swollen. A high number of soot particles 1). Retrieved January 23, 2014 from: http://www.fao.org/ag/againfo/themes/en/ and tar aerosols containing BaP adhere in the casing, mainly during meat/home.html. the first 5 days of smoking. Only a small amount of soot particles Fretheim, K. (1976). Carcinogenic polycyclic hydrocarbons in Norwegian smoked e migrates into the product, through the high size pores and the meat sausages. Journal of Agricultural and Food Chemistry, 24 (5), 976 979 . Fromberg, A., Højgård, A., & Duedahl-Olesen, L. (2007). Analysis of polycyclic aro- viscous fat. In contrast, synthetic casings have a smooth matic hydrocarbons in vegetable oils combining gel permeation chromatog- morphology and low porosity (16.6%). The fat remains inside the raphy with solid-phase extraction clean-up. Food Additives & Contaminants, 24 , e product They present a dry, hard external surface with less af finity 758 767 . García-Falcon, M. S., & Simal-G andara, J. (2005). Polycyclic aromatic hydrocarbons for soot particles, whose possibility of penetrating into the product in smoke from different woods and their transfer during traditional smoking is hindered by the small pore size of the casing and dry surfaces. For into chorizo sausages with collagen and tripe casings. Food Additives and Con- all these reasons, a high BaP content was found in the casings, but it taminants, 22 , 1 e8. Gomes, A., Santos, C., Almeida, J., Elias, M., & Roseiro, L. C. (2013). Effect of fat was only detected inside the meat products stuffed in natural content, casing type and smoking procedures on PAH contents of Portuguese casing (1.71 ± 0.15 mg/kg). No BaP was found inside the meat traditional dry fermented sausages. Food and Chemical Toxicology, 58 , products stuffed in synthetic casings. Finally synthetic casings 369 e374 . Guillen, M. D., Sopelana, P., & Palencia, G. (2004). Polycyclic aromatic hydrocarbons contribute to reducing BaP levels in smoked meat products. and olive pomace oil. Journal of Agricultural and Food Chemistry, 52 , 2123 e2132 . Hitzel, A., P ohlmann,€ M., Schw agele,€ F., Speer, K., & Jira, W. (2013). Polycyclic aromatic hydrocarbons (PAH) and phenolic substances in meat products smoked Acknowledgements with different types of wood and smoking spices. Food Chemistry, 139 (1 e4), 955e962. The authors would like to thank the Food Technology, Photonics Ib anez,~ R., Agudo, A., Berenguer, A., Jakszyn, P., Tormo, M. J., & Sanch ez, M. J. (2005). Dietary intake of polycyclic aromatic hydrocarbons in a Spanish population. Microscopy and Image Processing and Scanning Electron Micro- Journal of Food Protection, 68 , 2190 e2195 . scopy services of the Scienti fic-Technical Services of the University Jira, W., Ziegenhals, K., & Speer, K. (2006). PAH in smoked meat products. Studies of Oviedo for the assistance provided, specially to Dra. Amanda Laca according to the new EU requirements (original title in German). Fleisch- e Perez, Dra. Marta Alonso Guervos and Dr. Alfredo Jesús Quintana wirtchaft, 10 , 103 106 . Ledesma, E., Rendueles, M., & Díaz, M. (2014). Benzo(a)pyrene penetration on a García. smoked meat product during smoking time. Food Additives and Contaminants, 31 , 1688 e1698 . Lodovici, M., Dolara, P., Casalini, C., Ciappellano, S., & Testolin, G. (1995). Polycyclic References aromatic hydrocarbon contamination in the Italian diet. Food Additives and Contaminants, 12 , 703 e713 . ~ Andree, S., Jira, W., Schwind, K. H., Wagner, F., & Schwagele, F. (2010). Chemical Lorenzo, J. M., Purri nos, L., Bermudez, R., Cobas, N., Figueiredo, M., & García safety of meat and meat products. Meat Science, 86 , 38 e48 . Fontan, M. C. (2011). Polycyclic aromatic hydrocarbons (PAH) in two Spanish ATSDR (Agency for Toxic Substances and Disease Registry). (1995). Toxicological traditional smoked sausage varieties: “Chorizo gallego” and “Chorizo de profile for polycyclic aromatic hydrocarbons . U.S. Department of Health and cebolla”. Meat Science, 89 , 105 e109 . Human Services-Public Health Service (cited 1995 august). 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Resultados

4.4 INFLUENCIA DEL TIPO DE TRIPA EN LAS PROPIEDADES DEL CHORIZO DURANTE EL AHUMADO: TEXTURA, COLOR Y CARACTERÍSTICAS ÓPTICAS

Los resultados obtenidos en el capítulo 4.3 indican que, a diferencia de las tripas naturales, y debido principalmente a su menor tamaño de poro y porosidad, las tripas de colágeno minimizan el flujo del contenido graso del chorizo hacia la superficie exterior, previniendo la captura y penetración en el producto de partículas procedentes del ahumado que contiene BaP y HAP cancerígenos.

Las tripas naturales han sido utilizadas desde hace más de 2000 años y numerosas entidades, como la Asociación Europea de Tripas Naturales para Embutidos (ENSCA), o la Asociación Europea de Tripas Naturales para Embutidos (INSCA), defienden sus propiedades para el desarrollo de productos cárnicos, destacando entre otros factores su arraigo a lo tradicional, su menor grosor, su resistencia a romper, o su dificultad para despegarse del producto (ENSCA, 2015; FEDECARNE, 2013; INSCA, 2015). Por otro lado, la creación de nuevas tripas artificiales ha sido necesaria para el desarrollo de la industria cárnica. Las tripas de colágeno fueron creadas por primera vez en Alemania, en el año 1925. Desde entonces han sido optimizadas, y su fabricación se consolida sobre los años 60 (Devro, 2015). Los productores de tripas artificiales defienden sus propiedades, su capacidad para homogeneizar la producción, el ahorro de tiempo productivo y en definitiva de costes industriales para el fabricante (Devro, 2015; Viscofan, 2015). La competencia entre los 2 sectores es muy elevada. Según datos del informe anual de Viscofan (líder mundial en la producción de tripas sintéticas), el tamaño mundial del mercado de venta de tripas para embutidos es mayor de 4,2 billones de euros (Viscofan, 2012). El mercado de tripas sintéticas está creciendo, pues el reparto global era de 70%-30% y 63%-37% para tripa natural y sintética, en 2011 y 2012, respectivamente, según Devro (Devro, 2013; Ryan, 2013) y al 58-42% y 50-50%, según Viscofan (Viscofan, 2012).

En el siguiente trabajo se determinaron y compararon científicamente la textura, el color, la humedad y la evolución morfológica de chorizos asturianos embutidos en tripa natural y de colágeno, mediante el estudio de 48 chorizos durante 11 días de ahumado. Mediante este trabajo se obtuvieron nuevas evidencias de la contaminación de los chorizos embutidos en tripa natural por las partículas de hollín que transportan HAP.

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Publicación: Texture, colour and optical characteristics of a meat product depending on smoking time and casing type. Situación: enviada para su evaluación a Food Chemistry (ELSEVIER).

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TEXTURE, COLOUR AND OPTICAL CHARACTERISTICS OF A MEAT PRODUCT DEPENDING ON SMOKING TIME AND CASING TYPE

Estefanía Ledesma a, b,* , Amanda Laca a, Manuel Rendueles a, Mario Díaz a

Corresponding author. Tel.: +34 985103439; fax: +34 985 103434.

E-mail address: [email protected] (M. Rendueles), [email protected] (M. Díaz).

ABSTRACT

Synthetic casings have recently been found to prevent the penetration of carcinogenic compounds in meat products during smoking, in contrast to natural casings. In this paper, physical characteristics of 48 chorizos encased in natural (ChN) and synthetic (ChS) casings, during 11 days of direct smoking, were compared by means of texturometry, colorimetry, moisture, Scanning Electron Microscopy and Fluorescence Stereo Microscopy analyses. The lightness (L*) of ChS is lower (p=0) than ChN. During 5 days, ChS presents higher hardness and browning index, and less redness (a*), yellowness (b*) and chroma than ChN (p<0.05). At 7 days, no differences (p>0.05) were found in a*, b*, hue angle or chroma. Except at 1 day, no differences (p>0.05) were found in their moisture. Physical characteristics were suitable in both cases, but synthetic casing reduces production time, prevents the penetration and accumulation of soot particles in mass cavities, and enables the standardization of chorizo.

Keywords: chorizo, smoked meat products, casing, colour, texture, microstructure.

1. Introduction

The consumption of meat and meat products, which contain important levels of protein, vitamins, minerals and essential micronutrients, is growing in developing countries. Meat

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processing provides the opportunity to add value, reduce prices, improve food safety and extend shelf life. However, unsuitable processing can lead to undesirable health effects and environmental impact (EC, 2013; FAO, 2013). Chorizo is a typical Spanish meat product. In northern Spain and other European countries, chorizo is exposed to direct smoking with the main aim of prolonging its shelf life and conferring sensorial characteristics. During non-controlled smoking, however, chemical contaminants are also formed, such as polycyclic aromatic hydrocarbons (PAH), dioxins, formaldehyde, nitrogen and sulphur oxides (relevant in the formation of nitrosamines, for instance) and even heavy metals (CAC, 2009). PAH have been found to be carcinogenic for humans (ATSDR, 2009).

In order to protect consumers from PAH contamination, laws and codes of best practices in food processing have been proposed. Regulation (EU) No. 835/2011 of 19 August 2011 (EC, 2011) has recently reduced the maximum permissible content in BaP (a marker of the presence of PAH) in smoked meat and smoked meat products from 5. 0 to 2.0 •g/kg in wet weight from 1/9/2014 on. The permissible sum of PAH4 (benzo(a)pyrene, benzo(a)anthracene, benzo(b)fluoranthene and chrysene) in these foods has been reduced from 30.0 to 12.0 •g/kg in wet weight from 1/9/2014 on. The “Code of practice for the reduction of contamination of food with (PAH) from smoking and direct drying processes” (CAC/RCP 68/2009) advocates the control of certain variables to prevent PAH contamination. A number of studies have been carried out to test these variables, including the kind of fuel (Hitzel, Pöhlmann, Schwägele, Speer, & Jira, 2013; Pöhlmann, Hitzel, Schwägele, Speer, & Jira, 2012; Stumpe- V!ksna, Bartkevics, Kukare, & Morozovs, 2008), smoking method (Pöhlmann et al., 2012; Pöhlmann, Hitzel, Schwägele, Speer, & Jira, 2013a; Škaljac et al., 2014; Varlet et al., 2007) , duration of smoking (Djinovic, Popovic, & Jira, 2008; Essumang, Dodoo, & Adjei, 2013; Ledesma, Rendueles, & Díaz, 2014), fat content (Pöhlmann, Hitzel, Schwägele, Speer, & Jira, 2013b), distance between the food and the heat source, position of the food in relation to the heat source (Pöhlmann et al., 2013b), temperature during smoking and direct drying (Pöhlmann et al., 2012), cleanliness and maintenance of equipment, and design of the smoking chamber and the equipment used for producing the smoke/air mixture (Pöhlmann et al., 2013a). Moreover as a post-smoking treatment, CAC (2009) advocates cleaning the product by rinsing or immersing it in water to remove soot and particles containing PAH from the surface of the food. It has been found that the greatest amount of BaP (and probably other PAH) is deposited in the casing of the meat product, subsequently migrating into the product (Andrée, Jira, Schwind, Wagner, & Schwägele, 2010; Djinovic et al., 2008; García-

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Falcón, & Simal-Gándara, 2005; Ledesma et al., 2014; Santos, Gomes, & Roseiro, 2011). Some authors have reported that the use of different types of casing influences the PAH content of meat products (Filipovic & Toth, 1971; Toth, 1973). Recent studies (Gomes, Santos, Almeida, Elias, & Roseiro, 2013; Ledesma et al., 2015; Škaljac et al., 2014 ) have concluded that the use of synthetic instead of natural casings contributes to reducing PAH levels in smoked meat products. The causes of this effect have been attributed to differences in the morphology and porosity of natural (16.63%) and synthetic (66.84%) casings (Ledesma et al., 2015).

The aim of the present study was to compare the physical characteristics (texture, colour, moisture and morphology) of chorizos encased in natural and synthetic casings during direct smoking time in order to determine which casing is better for producing high quality chorizos safe for human consumption.

2. Materials and methods

2.1 Preparation of meat products

Two kinds of meat products were prepared for this study: chorizo encased in natural casing (ChN), and chorizo encased in synthetic casing (ChS). Twenty-four chorizos of each kind were manufactured at the El Hórreo Healthy Food S.L. meat company using pork loin (46.8%), pork jowl (46.8%), salt (1.8%), garlic (1%), sweet or spicy paprika (2%) and herbs (1.6%), all of which were minced, mixed and encased. Antioxidants such as sodium citrate or sodium ascorbate, colourings such as carmine, emulsifiers such as sodium triphosphate, food preservatives such as sodium nitrite and other food additives were used to ensure the microbiological stability and quality of the foodstuff. Nutritional analysis of the product was carried out by the Principality of Asturias Meat Industry Association Technological Centre for Supporting Innovation (Spanish acronym, ASINCAR), providing the following results: 31.41% moisture content, 22.98% protein, 37.94% fat, 2.85% carbohydrates, 4.82% ash and pH 5.2.

A batch of chorizo was processed with the aim of reproducing industrial manufacturing conditions. The minimum amount of chorizo considered as an industrial production volume is 50 kg. If an insufficient amount of chorizo is introduced in the smoking chamber, it could be exposed to an excess of smoke. Thus, 50 kg of raw chorizo ingredients were minced, mixed, marinated

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and stuffed to obtain 200 strings of chorizo, of which 12 strings (containing 48 chorizos) were analyzed. All the selected samples were placed in the same position in the direct smoking chamber at the same distance from the smoke source, namely 10 m. Only oak wood was used for direct smoking of the chorizos. Temperature and humidity parameters in the direct smoking chamber ranged between 7-17 ºC and 49-100 % respectively, depending on weather conditions and intermittent smoking. One string of each kind of chorizo was sampled at the following smoking times: 0, 1, 4, 5, 7 and 11 days, between 7 th and 18 th November. Once smoked, the chorizos were hung from a cardboard tube inside a box, avoiding any contact of the materials.

2.2 Apparatus

2.2.1 Fluorescence Stereo Microscope

The external and internal surfaces (once the casing was removed) of the chorizos were studied using a Fluorescence Stereo Microscope (Leica M205 FA, Wetzlar, Germany) to determine the microscopic morphology of the samples.

2.2.2 Texture measurement

In this study, the hardness of ChN and ChS during smoking time (0, 1, 4, 5, 7, and 11 smoking days) was analyzed in triplicate. Whole chorizo samples were placed on the heavy duty platform of the texture analyser (TA.XTPlus, Hamilton, MA, USA) and the arm of the texture analyser moved down to penetrate the product before returning to its initial position. A TA-18 rounded end probe with a 1/2" diameter stainless steel ball was used for this purpose.

2.2.3 Colour measurement

Chorizo colour measurements (L*, a*, b* CIELAB values) were carried out using an UltraScan VIS spectrophotometer (HunterLab, Reston, VA, USA). The instrument was standardized using a white title and calibration was tested using a green ceramic plate . The Hunter L*, a*, and b* values correspond to lightness, black (-l) or white (+l), greenness (-a) or redness (+a), and blueness (-b) or yellowness (+b), respectively. The colour measurements were performed on chorizos at room temperature (20 ± 2 ºC). Three different samples of each kind of chorizo (ChN and ChS) were selected after different days of smoking (0, 1, 4, 5, 7 or 11) and colour parameters were measured 30 times for each chorizo per smoking day.

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Total colour change (•E; Eq. (1)), hue angle (Eq. (2)), chroma (saturation index; Eq. (3) ), and browning index (BI; Eq. (4)) were calculated using Hunter L, a, and b values (Bozkurt & Bayram, 2006; Homco-Ryan et al., 2004; Laca, Sáez, Paredes, & Díaz, 2010; Maskan, 2001) as:

Eq.(1)

Hue angle= Eq. (2)

Chroma= Eq. (3)

Eq. (4) where:

2.2.4 Scanning electron microscopy (SEM)

A Jeol 6100 scanning electron microscope operating at 30 kV was used to determine the microscopic morphology of the samples. Chorizos were divided in two parts for SEM analysis: the casings, and the chorizos without casings. Samples were pretreated by means of freezing to 193.15 K for 4 hours followed by freeze-drying in a lyophilizer (Telstar-Cryodos, Spain) for 1 day.

2.2.5 Determination of moisture content

Moisture content was determined by gravimetry, in triplicate, during 0, 1, 4, 5, 7 and 11 days of smoking of ChN and ChS. Twenty g of thick grain sea sand (ref. 211161, Panreac, Spain) were weighed in a stainless steel mortar with its pestle The mortar was then kept in a vacuum desiccator for 30 minutes until its weight value was constant. Subsequently, approximately 6 g of sample were weighed into the mortar and the sea sand and chorizo sample were mixed with the pestle. The mortar was placed in an oven (Memmert, Germany) at 105±2ºC for 5 hours. The mortars were then allowed to cool in a vacuum desiccator at room temperature for 30 minutes before finally being weighed.

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2.2.6 Statistical analyses

Data were analysed by running t-tests to compare means, at a significance level of p<0.05. Previously, F-tests were carried out at a significance level of p<0.05 to compare standard deviations. Standardized skewness and standardized kurtosis were used to assess whether the samples came from normal distributions. Multiple range tests were applied between chorizo samples stuffed in the same kind of casing during smoking time in order to determine which means were significantly different from which others. The software used was Statgraphics Plus 3.1.

3. Results and discussion

3.1 Texture measurement

Figure 1 shows the hardness results for ChN and ChS during smoking time (0, 1, 4, 5, 7, and 11 smoking days). As can be seen in this figure, the hardness of both types of chorizos presents a similar trend: hardness increases during drying. However, the hardness of ChN and ChS was found to be different (p<0.05) from the moment when the products were stuffed (before smoking) to 7 days of smoking. The hardness of ChS (from 255.24g ± 9.60 to 999.27 ± 24.13) was always higher than that of ChN (from 66.57g ± 1.14 to 851.62g ± 22.93). However, no statistical significant differences (p>0.05) were found between the two groups at 11 days of smoking.

Figure 1. Hardness (g) texture profile analysis of chorizos stuffed in natural (ChN) and synthetic (ChS) casings during smoking time.

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According to our results, higher values of hardness were found in more dried Spanish “chorizo de Pamplona” after about 30 days of fermentation, smoking and drying processes (Gimeno, Ansorena, Astiasarán, & Bello, 2000). Softness can be a positive attribute in some types of chorizo (Melendo, Beltran, Jaime, Sancho, & Roncales , 1996), while in others (like ‘‘chorizo de Pamplona”) it can be considered a defect (Gimeno , Astiasarán, & Bello, 1999). In fact, the Spanish chorizo studied in this paper (chorizo from northern Spain) is used as an ingredient in typical Spanish stews with a long cooking time, such as “fabada” (about 3 hours). For this reason, this kind of chorizo should not be too hard before cooking so as to allow its ingredients to provide a good taste to the final dish. Synthetic casing could hence reduce processing time to obtain the same hardness as that of ChN.

3.2 Colour analysis

Figure 2 shows the lightness (L*) (2A), redness (a*) (2B), yellowness (b*), (2C) browning index (BI) (2D), hue angle (2E), chroma (2F) and total colour change (•E) (2G) of the analyzed Asturian ChN and ChS during smoking time (0, 1, 4, 5, 7 and 11 days).

There were no differences in the ingredients or preparation of the ChN and ChS, except for the use of different types of casing (natural and synthetic). However, as Figure 2 shows, the two types of casings produce differences in chorizo colour from the moment the products are stuffed. Before smoking, synthetic casings produced significantly (p<0.05) less lightness (L*: 44.01±1.29), redness (a*: 33.09±0.96), yellowness (b*: 32.50±0.81) and chroma (46.38±1.21) and a higher browning index (170.46±2.53) in unsmoked chorizo than natural casings (L*: 47.14±0.90; a*: 33.92±0.57; b*: 33.16±0.45; BI: 159.90±4.69; CH: 47.44±0.22). In contrast, no significant differences (p>0.05) were found in the hue angle of either type of chorizo before smoking.

During 1 to 11 days of smoking, ChN always continues to show more lightness (p=0 from 0 to 7, p= 0.000012 at 11 smoking days, from 39.18±0.32 to 29.73±2.55) than ChS (from 35.39±0.56 to 26.41±0.68). Redness showed more variable behaviour in chorizos stuffed in the two types of casing during smoking. At the beginning of smoking (1 day), no differences in redness (p>0.05) were found in the two types of chorizos. After 4 and 5 days of smoking, ChN showed higher redness (p<0.05) than ChS, a finding which was also observed (p>0.05) at 7 days of smoking. Finally, the opposite effect appeared after 11 days of smoking, when a higher a* was found in ChS (19.99±0.17) than in ChN (18.84±0.75). Likewise, from 1 to 5 days of smoking, the yellowness

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of ChN was higher than ChS, with no differences (p>0.05) finally being found between the two groups from 7 days of smoking on, when b* dropped to 12 after 11 days. From 4 to 11 days of smoking, the BI of ChS was higher (p=0) (from 148.24±11.80 to 111.52±5.06) than that of ChN (from 133.36±15.32 to 94.70±4.21). From 1 to 5 days of smoking, the HA and CH of ChN was found to be higher (p<0.05) than that of ChS. Like yellowness, no differences were found (p>0.05) in HA from 7 days of smoking on in the two kinds of chorizos. Like redness, no differences were found (p>0.05) in the CH of the two types of chorizos at 7 days of smoking. The opposite effect was found at 11 days of smoking, when the CH of ChS was significantly (p<0.05) higher (24.03±1.16) than that of ChN (22.13±0.97). Finally, both kinds of casings produced differences in the colour of smoked chorizo. Synthetic casing gives a darker colour at the same smoking time, maintaining redness and affording the product a “more cooked” appearance. Differences in the colour of meat products stuffed in different casings were also found by Pöhlmann et al. (2013b).

As Figure 2G shows, the highest total colour change (!E) in ChN and ChS was produced between before (0 days) and after 4 days of smoking, probably because the product is modified from “unprocessed” to “processed” and con stitutes the most intense change. The total colour change (!E) was less pronounced from 1 to 4 days of smoking, probably because of the occurrence of a weekend between these days, when stoking of smoke is lower. From 4 to 7 days of smoking, the product is already cooked and intense changes are not produced, obtaining a very low increase of !E. In fact, manufacturers used to define 5 -6 days of smoking as the end of processing. Finally, !E increased from 7 to 11 days, when the product is “over cooked”.

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Figure 2. Lightness (L*) (2.a), redness (a*) (2.b), yellowness (b*) (2.c), browning index (BI) (2.d), hue angle (2.e), chroma (2.f) and total colour change (•E) (2.g) of Asturian chorizo stuffed in natural (ChN) and synthetic (ChS) casings during smoking time.

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Figure 2. (continued).

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Figure 2. (continued).

3.3 Moisture content study

Table 1 shows the moisture content results for ChN and ChS during smoking time (0, 1, 4, 5, 7 and 11 days). As can be seen in this table, the moisture content of both kinds of chorizos shows a similar trend. Before smoking, both kinds of chorizos have the same moisture content (47.22% ±3.36), as they have the same ingredients. From 4 days of smoking on, ChN always shows a higher moisture content than ChS, although no significant differences (p>0.05) were found between ChN and ChS. Furthermore, the moisture content stabilized (p>0.05) after 5 days of smoking in each type of chorizo.

Table 1. Moisture profile analysis of chorizo stuffed in natural (ChN) and synthetic (ChS) casing during smoking time (n=3). Smoking time (days) 0 1 4 Chorizo and “p” ChS ChN p ChS ChN p ChS ChN p Average moisture 1 0.04 0.48 47.22 47.22 44.29 37.80 33.02 35.51 content (%) S.D. 3.36 3.36 3.04 2.14 4.26 3.49 Smoking time (days) 5 7 11 Chorizo and “p” ChS ChN p ChS ChN p ChS ChN p Average moisture 0.14 0.09 0.40 28.21 32.38 27.36 31.29 26.45 content (%) 25.67 2.97 2.62 2.49 1.26 0.88 1.12 S.D.

ChS: Chorizo stuffed in Synthetic casing ChN: Chorizo stuffed in Natural casing p: probability of significant differences between 2 samples 3.4(p<0.05: SEM thereanalysis is a significant difference between the two means). S.D.: Standard deviation n: number of analyzed samples

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Figure 3 shows the SEM images for ChN and ChS during smoking time. Figure 3.a and 3.b show the physical structure of raw chorizo mass before it is introduced in a casing. As these figures show, the unsmoked raw mass of chorizo is a non-homogeneous or non-compact material, full of large cavities. Figure 3.c shows the external surface of ChN after 3 days of smoking, where soot particles and small holes can be seen on the casing surface. The holes could be cavities on a fat or soot layer. Figures 3.d to 3.g show the physical structure of external (3.d, 3.f) and internal (3.e, 3.g) depths of slices of 5-day-smoked chorizo stuffed in natural (3.d, 3.e) and synthetic (3.f, 3.g) casings. As these figures show, after 5 days of smoking the smoked chorizo mass has become a more homogeneous and compact material, with no cavities, the opposite to what is observed in Figures 3.a and 3.b. The internal depth of ChN (3.e) and ChS (3.g) is smoother and less irregular than the unsmoked raw mass (3.a and 3.b). During smoking, the water content in chorizo probably starts to flow up from the internal depth of the product across the large cavities present in the raw material. The components of the inner mass of both types of chorizo start to blend together and cavities get smaller until disappearing. This fact could explain the finding that the water exiting both kinds of chorizos and their moisture content is similar. As result of drying, the roughness of the chorizo surface probably decreases from the external to the internal depth of the product, where more protuberances were found.

Figures 3.h and 3.i show the structure of natural and synthetic unsmoked casings (before processing), respectively. As can be seen in Figure 3.h and 3.i, a higher number of small, elongated white objects were found in natural (3.h) than in collagen (3.i) casings. The casings were found to present a different morphology. According to previous results (Ledesma et al., 2015 ), these objects were identified by elemental analysis using SEM as crystals of salt (42.75% Na and 57.25% Cl). Natural casings were found to be wrinkled, while synthetic casings were found to be smooth and formed by clear collagen fibres. Figures 3.j and 3.k show the SEM images of ChN and ChS casings after 5 days of smoking, respectively. Soot particles from the smoke damage and penetrate the natural casing (3.j), while they seem to be deposited on the synthetic casing (3.k), without penetrating it.

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a b c

d e

f g

i h

Figure 3. SEM images of chorizo before processing (a,b), external surface of 3-day-smoked chorizo stuffed in natural casing (ChN) (c), external and internal depths of 5 -day- smoked chorizo stuffed in natural (d,e) and synthetic (ChS) (f,g) casings. Natural and synthetic casings before (h,i) and after (j,k) 5 days of smoking. The scale of Figures “a”, “d”, “e”, “g” and “k” is 500 •m; the scale of Figures “b”, “f”, “h” and “i” is 100 •m. The scale of Figures “c” and “j” is 10 •m and 200 •m, respectively.

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j k

Figure 3. (continued).

In previous studies (Ledesma et al., 2015), a higher degree of porosity was found in natural casings (66.84%) than in synthetic casings (16.63%). The wrinkled morphology of natural casing is more able to capture particles than synthetic casing. The fat content in chorizos flows through the natural casings, covering their external surface and making it sticky and wet. Soot particles slowly penetrate the internal parts of these meat products and migrate to the interior, preferentially through the lumps of fat. Finally, it can be stated that raw chorizo mass is full of cavities before smoking. During smoking, soot particles pass through the pores in natural casing. They can then pass through cavities in the raw ingredients and reach the internal parts of the product. At the same time, water exits the chorizo through these cavities, raw ingredients blend together and possible soot particles can be captured in the final compact mass.

3.4 Fluorescence Stereo Microscope analysis

Figures 4 and 5 show the general (without zoom) and enlarged (with zoom) external surface of ChN (left column, images a, c, e, g and i) and ChS (right column, images b, d, f, h and j) from 0 to 10 days of smoking, with the exception of Figures 5.a and 5.b, which respectively show unprocessed natural and synthetic casings (without raw ingredients stuffed inside them). Natural and synthetic casings produce different behaviour in chorizos throughout the manufacturing process, before and during each smoking day. As Figures 4.a and 4.b show, the chorizo mass seems to exit natural casings (4.a), while it remains inside synthetic casing (4.b). As can be seen in Figures 4.c, 4.e, 4.g and 4.i, the external surface of ChN gets progressively stickier and covered in

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fat during 1, 4, 5 to 10 days of smoking time, respectively. The chorizo seems to swellb up when flat flows across the casings. In contrast, the external surface of ChS (4.d, 4.f, 4.h and 4.j) is always dry and the casing is clearly identified by the shiny plastic material. During smoking time, ChS seems to be wrinkled and clear lines are found in these chorizos (4.f, 4.i).

Figure 5.a shows that unprocessed natural casing is irregular. In contrast, Figure 5.b shows that synthetic casing is full of aligned, elongated collagen fibres. After only 1 day of smoking, small soot particles can be found on the sticky surface of ChN (Figure 5.c). The production of these soot particles and tar aerosols during meat product smoking and pyrolysis of biomass at high temperatures (above 750ºC) produces contamination of meat products by carcinogenic PAH (Basu, 2010, Ledesma et al., 2015). With increasing smoking time, the number of soot particles was seen to increase and they also appeared to increase in size (Figures 5.e, 5.g, 5.i). In contrast, no fat was found on the dry surface of ChS and almost no soot particles were found on them either, even after 10 days of smoking (5.j). The elongated, lined fibres of collagen are found in the surface of ChS and wrinkles seem to get deeper with increasing smoking time. Areas where soot particles were found on the external surface of chorizos with casing were selected for analysis as shown in Figures 5.k and 5.l. Figures 5.k and 5.l show the external surface of ChN and ChS, respectively, when casings were removed. As can be appreciated in these figures, soot particles seem to cross the natural casing and are found when the casing was removed (5.k). In contrast, no soot particles were found when the synthetic casing was removed (5.l). The findings of this study are in agreement with previous research by the authors (Ledesma et al., 2015). Synthetic casing prevents the penetration of soot particles and carcinogenic compounds into meat products.

ChN ChS Smoking days a b

Figure 4. Fluorescence Stereo Microscope general images (no zoom) of chorizo before (a, b) and after the following smoking days: 1 (c,d), 4 (e,f), 5 (g,h), 10 (i,j). Pairs of images are chorizo stuffed in natural (ChN) and synthetic (ChS) casings, respectively. The scale of all images is 500 µm. 167

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ChN ChS

Smoking c d days

e f

g h 5

i j 10

a b Figure 4. (continued).

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Smoking natural casing synthetic casing days a b

ChN ChS c d

e f

g h

Figure 5. Fluorescence Stereo Microscope images of natural and synthetic casing before smoking (a, b)

(without zoom) and chorizo after the following days of smoking: 1 day (c,d), 4 days (e,f), 5 days (g,h) and 10 days (i,j and k,l).Pairs of images are chorizo stuffed in natural (ChN) and synthetic (ChS) casings, respectively. K and l images were taken from the external surface of ChN and ChS, once the casings were removed. The zoom factor of all images is 59, except for e (X43) and g (X21). The scale in all images is 500 µm. 169

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Smoking ChN ChS days

i j

10 l k

Figure 5. (continued)

4. Conclusions

The use of different types of casing (natural and synthetic) produces differences in the physical properties of chorizo during smoking. The texture, colour and moisture content values of chorizos stuffed in natural and synthetic casings were suitable in both cases. Over a period of 11 days of smoking, chorizos stuffed in synthetic casings showed darker colour, harder texture and drier mass than chorizos stuffed in natural casing. However, no significant differences (p>0.05) were found in the moisture content between the two groups. According to previous results, the structure of synthetic casing was found to prevent the accumulation and penetration of soot particles into the chorizo, while new evidence is linked to the capture of soot particles in the cavities of raw chorizo mass during drying. Finally, the use of synthetic casings in chorizo production could help to reduce processing time, prevent contamination by unhealthy products from smoking and achieve product standardization and homogenization, food properties demanded by customers and wholesalers.

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Acknowledgements

The authors would like to thank the Scientific-Technical Services at the University of Oviedo for the assistance provided.

References

ATSDR (Agency for Toxic Substances and Disease Registry). (2009). Case studies in environmental Medicine toxicity of Polycyclic Aromatic Hydrocarbons (PAH). Cited 2009 July 1. Retrieved May 11, 2014 from: http://www.atsdr.cdc.gov/csem/pah/docs/pah.pdf

Andrée, S., Jira, W., Schwind, K.H., Wagner, F., & Schwägele, F. (2010). Chemical safety of meat and meat products. Meat Science, 86, 38 –48.

Basu, P. (2010). Biomass Gasification and Pyrolysis Practical design and theory. Burlington (MA): Elsevier, (Chapter 4).

Bozkurt, H., & Bayram, M. (2006). Colour and textural attributes of sucuk during ripening. Meat Science, 73, 344 –350.

CAC (Codex Alimentarius Commission). (2009). Code of practice for the reduction of contamination of food with polycyclic aromatic hydrocarbons (PAH) from smoking and direct drying processes (CAC/RCP 68/2009). (Cited 2009). Retrieved May 11, 2014 from: http://www.codexalimentarius.org/standards/list-of standards/en/?provide=standards&orderField=fullReference&sort=asc&num1=CAC/RCP

Djinovic, J., Popovic, A., & Jira, W. (2008). Polycyclic aromatic hydrocarbons (PAHs) in different types of smoked meat products from Serbia. Meat Science, 80, 449 –456.

Essumang, D.K., Dodoo, D.K., & Adjei, J.K. (2013). Effect of smoke generation sources and smoke curing duration on the levels of polycyclic aromatic hydrocarbon (PAH) in different suites of fish. Food and Chemical Toxicology, 58, 86 –94.

EC. (2011). European Union Commission (EC) (Internet). Regulation Nº 835/2011 of 19 August 2011 amending Regulation (EC) No 1881/2006 as regards maximum levels for polycyclic aromatic

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hydrocarbons in foodstuffs (EC, No 835, 2011) (Cited 2011 August 20). Available from: http://eur- lex.europa.eu/LexUriServ/LexUriServ.do?uri=OJ:L:2011:215:0004:0008:EN:PDF

EC. (2013). European Commission decision Horizon 2020 Work Programme 2014 – 2015. Part 9. Food security, sustainable agriculture and forestry, marine and maritime and inland water research and the bioeconomy. Safe food and healthy diets and sustainable consumption. SFS-15- 2014: Proteins of the future (pp. 26-27). (Cited 2013 December 10). Retrieved May 11, 2014 from: http://ec.europa.eu/research/participants/data/ref/h2020/wp/2014_2015/main/h2020- wp1415-food_en.pdf

Filipovic, J., & Toth, L. (1971). Polycyclische Kohlenwasserstoffe in geräucherten jugoslawischen Fleischwaren. Fleischwirtschaft, 51, 1323 –1325.

FAO. (2013). Food and Agriculture Organization of United Nations. Agriculture and Consumer Protection Department. (2013). Meat & Meat Products. Meat consumption (Cited 2013 March 1). Retrieved April 28, 2014 from: http://www.fao.org/ag/againfo/themes/en/meat/background.html

Gimeno, O., Astiasarán, I., & Bello, J. (1999). Influence of partial replacement of NaCl with KCl and CaCl2 on texture and colour of dry fermented sausages. Journal of Agricultural and Food Chemistry, 47, 873 –877.

Gimeno, O., Ansorena, D., Astiasarán, I., & Bello, J. (2000). Characterization of chorizo de Pamplona: instrumental measurements of colour and texture. Food Chemistry, 69, 195 –200.

Gomes, A., Santos, C., Almeida, J., Elias M., & Roseiro, L.C. (2013). Effect of fat content, casing type and smoking procedures on PAHs contents of Portuguese traditional dry fermented sausages. Food and Chemical Toxicology, 58, 369 –374.

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Homco-Ryan, C. L., Ryan, K. J., Wicklund, S. E., Nicolalde, C. L., Lin, S., McKeith, F. K., & Brewer, M.S. (2004). Effects of modified corn gluten meal on quality characteristics of a model emulsified meat product. Meat Science, 67, 335 –341.

Laca, A., Sáenz, M.C., Paredes, B., & Díaz, M. (2010). Rheological properties, stability and sensory evaluation of low-cholesterol mayonnaises prepared using egg yolk granules as emulsifying agent. Journal of Food Engineering, 97, 243 –252.

Ledesma, E., Rendueles, M., & Díaz, M. (2014). Benzo(a)pyrene penetration on a smoked meat product during smoking time. Food Additives and Contaminants, 31, 1688-1698.

Ledesma, E., Rendueles, M., & Díaz, M. (2015). Characterization of natural and synthetic casings and mechanism of BaP penetration in smoked meat products. Food Control, 51, 195-205.

Maskan, M. (2001). Kinetics of colour change of kiwi fruits during hot air and microwave drying. Journal of Food Engineering, 48, 169 –175.

Matulis, R. J., Floyd, K. M., Sutherland, J. W., & Brewer, M. S. (1995). Sensory characteristics of frankfurters as affected by salt, fat, soy protein and carrageenan. J. Food Science, 60, 48 –54.

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Santos, C., Gomes, A., & Roseiro, L.C. (2011). Polycyclic aromatic hydrocarbons incidence in Portuguese traditional smoked meat products. Food and Chemical Toxicology, 49, 2343 –2347.

Škaljac, S., Petrovi!, L., Tasi!, T., Ikoni!, P., Jokanovi!, M., Tomovi!, V., Džini!, N., Šoji!, B., Tjapkin, A., & Škrbi!, B. (2014). Influence of smoking in traditional and industrial conditions on polycyclic aromatic hydrocarbons content in dry fermented sausages (Petrovská klobása) from Serbia. Food Control, 40, 12 –18.

Stumpe- V"ksna , I., Bartkevics, V., Kukare, A., & Morozovs, A. (2008). Polycyclic aromatic hydrocarbons in meat smoked with different types of wood. Food Chemistry, 110, 794 –797.

Toth, L. (1973). Einfluss verschiedener Verfahren des Räucherns und Grillens auf den Gehalt von Fleisch und Fleischerzeugnissen an cancerogenen Stoffen. DFG Abschlussbericht: Teil I [Influence of different smoking and grilling methods on the carcinogenic substances content of meat and meat products]. DFG. Final Report: Part I (“title in German”).

Varlet, V., Serot, T., Knockaert, C., Cornet, J., Cardinal, M., Monteau, F., et al. (2007). Organoleptic characterization and PAH content of salmon (Salmo salar) fillets smoked according to four industrial smoking techniques. Journal of the Science of Food and Agriculture, 87 (5), 847 – 854.

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4.5 CONTAMINACIÓN DE PRODUCTOS CÁRNICOS POR HAP DURANTE EL AHUMADO: PROCESOS Y PREVENCIÓN.

Este apartado de la memoria realiza una revisión sobre el proceso tecnológico de ahumado de productos cárnicos y el control de la contaminación por HAP durante el mismo. En esta revisión se narra la historia del ahumado, buscando las primeras referencias históricas de aplicación de este proceso tecnológico alimentario y el avance de las técnicas hasta la actualidad. Así mismo, se describen los distintos tipos de ahumado, directo e indirecto, utilizados actualmente en la industria cárnica. Por otro lado se define la composición del humo y su objetivo en el ahumado de productos cárnicos. Se describe la formación química de HAP durante el ahumado y los distintos mecanismos mediante los cuales estos compuestos llegan a los productos en la industria cárnica, contaminándolos con sustancias cancerígenas. Se realiza una revisión de las distintas regulaciones internacionales publicadas y aplicadas durante los últimos 10 años para proteger a los consumidores sobre la ingesta de HAP en la dieta, en concreto, mediante el consumo de productos cárnicos. Estas regulaciones determinan la concentración máxima permitida de estos compuestos en los alimentos. Se describen y comparan los distintos métodos, clásticos y modernos, de pretratamiento de muestras y determinación analítica utilizados por la comunidad científica para identificar y cuantificar la presencia de estos compuestos en los productos cárnicos.

La parte central está enfocada en 2 apartados. Por un lado, en la revisión de la presencia en la actualidad de HAP en productos cárnicos ahumados a lo largo del mundo, y por otro lado, en las 10 variables del proceso tecnológico de ahumado que, de acuerdo al Codex Alimentarius Code CAC/RCP68/2009, se deben controlar para minimizar y prevenir la contaminación de HAP en alimentos ahumados y que por lo tanto han sido estudiadas en profundidad por la comunidad científica. Finalmente la revisión concluye con la comparación de todas las variables, seleccionando las que tienen más relevancia de acuerdo a la comunidad científica y evaluando la coherencia de los límites de HAP en productos cárnicos establecidos por la reglamentación.

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Publicación: Contamination of meat products during smoking by polycyclic aromatic hydrocarbons: Processes and Prevention.

Situación: Enviado a la revista Food Control (Elsevier).

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CONTAMINATION OF MEAT PRODUCTS DURING SMOKING BY POLYCYCLIC AROMATIC HYDROCARBONS: PROCESSES AND PREVENTION.

E. Ledesma a,b , M. Rendueles a & M. Díaz a

Corresponding author: Tel.: +34 985103439; fax: +34 985 103434.

E-mail address: [email protected] (M. Rendueles), [email protected] (M. Díaz).

ABSTRACT

Meat products may be contaminated by carcinogenic polycyclic aromatic hydrocarbons (PAH) during smoking. The maximum PAH content allowed in meat products has recently been reduced by EU Regulation No 835/2011. Codex Alimentarius Commission code of practice CAC/RCP 68/2009 specifies the 10 variables that must be controlled in order to minimize and prevent PAH contamination of meat products during smoking. Each variable has been separately studied by several researchers. The aims of this paper are to describe PAH contamination of meat products during smoking, review recent data on PAH in meat products, and compare the influence of the different variables involved in smoking. PAH limits are met in the majority of meat products reported worldwide, but are still greatly exceeded in others. Three variables seem to have a greater effect in reducing PAH content: smoke generation temperature, type of casing, and smoking method (direct or indirect). A smoke generation temperature below 600°C must be applied to prevent the formation of PAH. The use of synthetic instead of natural casings prevents PAH from penetrating inside the products. Indirect smoking systems (i.e., friction or liquid smokes) can highly reduce the PAH content of meat products. Finally, meat product smoking can be adapted to prevent PAH contamination and comply with the new European limits.

Keywords: Polycyclic aromatic hydrocarbons (PAH), meat products, smoking process, CAC/RCP68/2009, contamination, control.

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1. Introduction

Meat and meat products are high economic interest foodstuffs, constituting the most valuable livestock products. World meat production is forecast to double by 2050. In 2015, the average meat consumption per capita worldwide is expected to be 41.3 kg/year. The projected consumption in developing and industrial countries will be 31.6 kg/year and 95.7 kg/year, respectively (FAO, 2013, 2014). Meat has played a crucial role in human evolution and is an important component of a healthy, well-balanced diet due to its nutritional richness (De Castro Cardoso Pereira & Dos Reis Baltazar Vicente, 2013). From the nutritional point of view, meat ’s importance derives from its high biological value protein, containing all the essential amino acids, as well as highly bioavailable minerals, vitamins and micronutrients like iron, selenium, zinc and vitamin B12 (De Castro Cardoso Pereira & Dos Reis Baltazar Vicente, 2013; FAO, 2014; USDA/HHS, 2010; WHO/FAO, 2003). Offal meats like liver are also crucial sources of vitamin A and folic acid (Biesalski, 2005). However, the consumption of meat and meat products has been associated with an increased risk of certain chronic diseases such as colorectal cancer (CRC) (Alexander, Miller, Cushing, & Lowe, 2010; Alexander, Weed, Cushing, & Lowe, 2011; Aune et al., 2013; Biesalski, 2005; Corpet, 2011; Demeyer, Honikel, & De Smet, 2008; Ferguson, 2010; McNeill & Van Elswyk, 2012; WCRF/AICR, 2007; WHO/IARC, 2008; WHO/FAO, 2003; Wyness et al., 2011), the third most common cancer in men (746,000 cases, 10.0% of the total) and the second in women (614,000 cases, 9.2% of the total) worldwide (IARC, 2012). This association has been rejected by recent studies (McAfee et al., 2010), as there is a certain degree of inconsistency between observational and experimental data on red meat and cancer (Dragsted et al., 2014). More studies and reviews are needed (Kim, Coelho, & Blachier, 2013), while the prevention of the carcinogenic activity of meat products should be researched, especially in terms of controlling the processing of meat products. Processing increases the value of meat products, prices can be reduced and shelf life can be extended (FAO, 2014). However, the composition of meat can be damaged by non-desirable substances such as nitrosamines, polycyclic aromatic hydrocarbons (PAH), heterocyclic amines, biogenic amines and lipid oxidation products (Olmedilla-Alonso, Jiménez-Colmenero, & Sánchez-Muniz, 2013). Special attention has been paid to the smoking process because of the contamination of meat products by PAH. The carcinogenic, mutagenic and bioaccumulative capacities of PAH have been reported by the Food and Agriculture Organization of the United Nations (FAO), the World Health Organization (WHO), (WHO, 2006), the International Agency for Research on Cancer (IARC), the European Scientific

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Committee on Food (SCF), (SCF, 2002), the European Food Safety Authority (EFSA) and the US Environmental Protection Agency (EPA). The maximum PAH levels in meat products have recently been reduced by European Union (EU) Regulation No 835/2011. PAH contamination in smoked meat products can be controlled, maintaining the beneficial effects of smoking and preventing its undesirable effects. In particular, Codex Alimentarius Commission code of practice CAC/RCP 68/2009 specifies the 10 variables that need to be controlled to minimize and prevent carcinogenic PAH contamination of meat products during smoking (CAC, 2009). Each of these variables has accordingly been separately studied by several researchers.

The aims of this paper are to define the smoking process, describe the contamination of meat products with carcinogenic PAH during smoking, review recent data on PAH in meat products worldwide, compare the incidence and effect of the different smoking variables and, finally, evaluate the regulations.

1.1 History

Food smoking is one of the oldest food technologies used by humankind since the beginning of time (Tóth, 1982). Humans probably first hung meat over the fire as a means of protecting it from canines. Subsequently, the preservative action of smoke in prolonging the meat ’s “shelf life ” was probably discovered (Šimko, 2002, 2009) . The first evidence of smoking as a technological process dates back 90,000 years to Poland. The oldest smoking house was discovered by archaeologists in a Stone Age colony located in Zwierzymec, near Krakow (Möhler, 1978). The greatest amount of information on meat smoking dates from Roman cultures, especially in the book by Marcus Cato De Agri Cultura , dating from 160 BC (Leroy, Geyzen, Janssens, De Vuyst, & Scholliers, 2013; Mateo, Caro, Figueira, Ramos, & Zumalacarregui, 2009; Möhler, 1978; Zeuthen, 2007), and from the Middle Ages, in the book by Marx Rumpolt ’s “New Cookbook” , published in 1581 (Möhler, 1978). Some researchers claim that the Romans may have learned the technique of smoking meat from the Gauls and Celts. Europeans emigrants exported the method worldwide, including the Americas, South Africa and Australia (Leroy et al., 2013). Nowadays, with the exception of some African and Asian countries, where a cold chain has not been widely established (Ogbadu, 2014; FAO-Thiaroye, 2015), smoking is only used to give foodstuffs an enhanced, specific organoleptic profile, improving their flavor, color and smell, as these are properties widely demanded by consumers (Lorenzo, Purriños, García Fontán, &

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Franco, 2010; Šimko , 2002; Vaz-Velho, 2003). It has been proved that the profile of meat products (color, odor, and flavor) is enhanced by increasing the time the smoke is in contact with meat products (Pöhlmann, Hitzel, Schwägele, Speer, & Jira, 2013a). The alternatives to smoking as a meat preservation method include modern techniques such as controlled drying chambers, liquid smoke ( Kostyra & Bary!ko -Pikielna, 2006; Lingbeck et al., 2014; Theobald et al., 2012) and other methods for preserving fresh meat by means of refrigeration (such as chilling, freezing and superchilling), ionizing radiation, chemical preservatives and biopreservation, high hydrostatic pressure (HHP) and packaging methods like vacuum packaging, modified atmosphere packaging (MAP), active packaging (AP), antimicrobial packaging and hurdle technology (HT) (Zhou, Xu, & Liu, 2010).

2. Smoking methods

Since the beginning of traditional, uncontrolled burning of biomass, the technique of smoking food has been improved until became a food technology process. There are a number of ways of classifying smoking methods based on the temperature of the smoke, the location of smoke generation with respect to the position of the foodstuff, and the device used for generating smoke. In this paper, the different smoking methods are classified in two main groups: direct and indirect smoking.

2.1 Direct smoking methods

During direct smoking, smoke is produced in the same chamber where the meat is processed (CAC, 2009). Direct smoking methods mainly comprise traditional techniques. The traditional smoking method consists of direct thermal degradation of wood to produce smoke (Ahmad, 2003). This definition can be extended, as others kinds of fuels were erroneously used in the past all over the world. In fact, humans used to burn any kind of waste in bonfires to produce smoke, such as food waste (coconut shells, ears of corn, fruit stones, etc.) and even newspapers or furniture (wood treated with paint) (CAC, 2009). Various unhealthy compounds are formed if the correct fuel is not used, especially PAH. Direct smoking methods can be classified according to the temperature of the smoke.

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2.1.1 Traditional cold smoking

During cold smoking, wood is burned and smoke is produced. Meat products are hung from shelves placed above the hearth, located in a grilled floor through which the smoke passes. Smoking chambers are usually large. When burning finishes, the fire is not poked and the smoke cools. According to different authors, the required temperature in a smoking chamber to achieve cold smoking conditions should be below 20°C (Möhler, 1978), between 15 -25°C ( Šimko , 2009; Woods, 2003), or below 30°C (Ahmad, 2003).

Raw hams (Möhler, 1978; Šimko , 2009), heat fermented untreated products like salami (Šimko , 2009) and other stuffed meat products, like chorizo, are usually produced by cold smoking.

2.1.2 Traditional hot smoking

2.2 Indirect smoking methods

Indirect smoking comprises a number of new methods that help reduce PAH contamination of meat products. These are summarized below.

2.2.1 Smoke produced by a friction generator

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friction smoke generation, smoke evacuation and drying. These steps are repeated in cycles, the number and duration of which depend on the type of meat product. Typical smoke generation values are 20-second intervals of continuous friction of a gearwheel with wood followed by a pause of between 70 and 175 seconds. During this process, a temperature range between 180 and 380°C is reported by Varlet et al. (2007). The process, adapted for the production of a typical Spanish meat product called chorizo, includes 5 cycles of 30 minutes of preheating (150 minutes), reducing the temperature from 18 to 2°C and the humidity from 95 to 90%, 6 hours of drying at 25°C and 90% humidity, 41 cycles, consisting of 10 minutes of smoking, 3 minutes of smoke evacuation, and 20 minutes of drying at 25°C and 90% humidity, and a final step of 3 cycles of 8 hours of drying (24 hours) at 80% humidity, decreasing the temperature from 23 to 18°C. According to Pöhlmann et al. (2013a), Frankfurter-type sausages are exposed to reddening for 10 min at 52°C, drying for 12 min at 56°C and friction smoking for 26 to 40 hours, followed by a final step of scalding at 75°C for 25 min. With friction smoke generation, operation time and wood requirements are reduced and production is controlled and optimized. Furthermore, meat industry safety is increased, as PAH production is very low (Pöhlmann et al., 2013a), workers ’ health is improved by preventing fire hazards, product weight losses are reduced, product flavor is enhanced by avoiding the concealing of the taste of the ingredients, product shelf life and quality are suitable and product standardization and homogenization is made possible.

2.2.2 Liquid smoke

Liquid smoke is a modern way of producing commercial smoked meat products more rapidly. It is more environmentally friendly and easier to apply than traditional smoking and allows good reproducibility of desired characteristics in the end product. Liquid smoke is produced by condensing wood smoke formed by the controlled, minimal oxygen pyrolysis of sawdust or wood chips. The wood is placed in large retorts where intense heat is applied, causing the wood to smolder (not burn), releasing the gases seen in ordinary smoke. These gases are quickly chilled in condensers, thus liquefying the smoke. The liquid smoke is then forced through refining vats and subsequently filtered to remove toxic and carcinogenic impurities containing PAH. Finally, the liquid is aged for mellowness (Lingbeck et al., 2014). In addition, liquid smoke exhibits antimicrobial activity against Listeria (Gedela, Gamble, Macwana, Escoubas, & Mariana, 2007; Martin et al., 2010; Messina, Ahmad, Marchello, Gerba, & Paquette, 1988), Escherichia coli (Van Loo, Babu, Crandall, & Ricke, 2012), Staphylococcus aureus and staphylococcal enterotoxins

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(Lingbeck et al., 2014; Taormina & Bartholomew, 2005). During its production, liquid smoke is filtered and subjected to fractionation and purification processes to remove toxic and carcinogenic particles and compounds. Therefore, its use is generally considered to be of less health concern than the traditional smoking process. However, the possibility of broader applications of smoke flavorings compared to conventional smoking has to be taken into account in safety assessments (EC No 2065/2003; Lingbeck et al., 2014).

2.2.3 Electrostatic smoking

In this type of smoking, the product is positioned in a continuous tunnel between live electrical wires that are charged to between 20 and 60 kV. Smoke passing through this system is charged according to its phase (smoke is a two-phase system, particulate and vapor), and smoke components can precipitate on the oppositely charged food surface (Vaz-Velho, 2003; Woods, 2003). The movement of gas and liquid in chambers has barely been studied (Pinilla, Díaz, & Coca, 1984). In order to ensure sedimentation of the smoke components on the surface of the product, the smoking step is usually followed by infrared irradiation (Möhler, 1978). This process helps to avoid PAH contamination of foodstuffs.

2.2.4 Other smoke generation technologies

Other well-known smoke generation technologies include steam, fluidization, touch and smoldering smoke generators. Steam smoke is produced by passing superheated steam through chopped wood, inducing pyrolysis. The resulting smoke passes through the smoking chamber, being cooled to 80°C (Prändl, Fisher, Schmidhofer, & Sinell, 1994; Tóth & Potthast, 1984). According to Müller (1982), the steam smoke generation temperature varies between 450 and 650°C. A fluidization smoke generator allows pyrolysis of wood shavings suspended in air that has been previously heated to 300-400°C. Pyrolysis is carried out within a reaction chamber and smoke and solid particles are separated on passing through a cyclone refiner (Nicol, 1960; Klettner, 1979; Tóth & Potthast, 1984; Prändl et al., 1994). The schematic representations of the different smoking generators are reported by Prändl et al. (1994). Smoldering, steam and touch smoke generation systems for the production of Frankfurter-type sausages are described in detail by Pöhlmann et al. (2012, 2013a). In these technologies, smoking conditions are highly controlled and optimized, including different smoke densities (light, medium, and intensive), temperatures of smoke generation (ranging between 300 and 520°C), ventilator speeds (750, 1500, and 3000

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rpm) and exposure times to smoke (from 3 to 40 minutes). The control of smoking conditions in these smoke generation technologies allows the prevention of final contamination of meat products with PAH (Pöhlmann et al., 2013a).

3. The smoking process

3.1 Aim and composition of smoke

Smoke composition is defined by the kind of fuel (wood), smoking conditions (temperature, time, humidity, air flow rate) and subsequent treatments of smoke. Up to 1100 chemical compounds have been identified in wood smoke (Wilms, 2000). Smoke composition has been described in detail (Ahmad, 2003; Hitzel et al., 2013; Stumpe- V•ksna, Bartkevics, Kukare, & Morozovs, 2008). Wood smoke is composed by over 400 volatile components comprising 48 acids, 22 alcohols, 131 carbonyls, 22 esters, 46 furans, 16 lactones, 75 phenols, and some 50 miscellaneous compounds (Woods, 2003). The main components of smoke condensates are water (82.42%), tar (4.81%), residue (4.21%), extracts from activated charcoal (4.08%), acetic acid and acids of higher molecular weight (1.71%), methanol (0.96%), ketones (0.67%), aldehydes of highe molecular weight (0.57%), formic acid (0.38%), formaldehyde (0.12%) and phenols (0.07%).

Table 1 (Ahmad, 2003 ; Guillén & Manzanos, 2002; Kostyra & Bary!ko -Pikielna, 2006; Möhler, 1978; Ojeda, Barcenas, Pérez-Elortondo, Albisu, & Guillén, 2002; Woods, 2003) summarizes the effects and function of the different components of smoke during meat processing, as described by Möhler (1978). The chemical compounds of smoke are organized in 20 groups. However, these can be divided in 2 main groups depending on their desirable or non-desirable effects in the end product. For instance, Thymol, formaldehyde, formic, acetic and benzoic acids, orthocresol, meta-cresol, para-cresol, guaiacol, methylguaiacol, cresol and xinelone have desirable bactericidal, antimicrobial, biocidal, fungicidal and preservative effects. Guaiacol, vinilguaiacol and butyric, valerianic, caproic, enanthic, caprylic, and pelargonic acids confer a good smell on products. During the first days of ripening, the coloring of meat products is mainly caused by the nitrogenous compounds present in meat in combination with myoglobin, resulting in the desired color pigment (Bozkurt & Bayram, 2006). Coloring of meat products is mainly caused by the well-known, non-enzymatic Maillard browning.

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Table 1. Chemical composition of smoke and technological effect. MAIN GROUPS OF COMPOUNDS IN SMOKE EFFECT ON MEAT PRODUCTS

COH 2 series compounds: xinelone. flavoring Aromatic compounds with Phenol Non desirable, bad taste one hydroxyl, phenol Phenol derivates: Desirable in low quantities Orthocresol, meta cresol, para -cresol Good effects in preservation, Xylenols smoke smell and color Thymol Strong antimicrobial attributes, biocide Fungicide effect Preservative Thyme aroma Group 5 Formaldehyde Desirable COH compounds Harden ing of natural casing (car bonyls): Aliphatic Connective tissue aldehydes Bactericide Food preservative Group 6 Benzaldehyde Desirable, COH compounds Bitter almond arom a (carbonyls): Aromatic aldehydes Group 7 Acetone and unsaturated long-chain Undesirable aroma CO compounds: Aliph atic compoun ds. ketones Group 8 Acetophenone Hay smell CO compounds: Hydrindenes Soft aroma Aromatic ketones Group 9 Formic acid and acetic acid. Desirable for: Color setting, COO Compounds: Alyphatic Good smell.Bactericidal effect carboxylic acids Butyric acid, valerianic acid, caproic acid, Smell enanthic acid, caprylic acid, pelargonic acid. Unsaturated carboxylic acid (crotonic and Desirable for color tiglic) No desirable for taste

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Table 1. (continued). MAIN GROUPS OF COMPOUNDS IN SMOKE EFFECT ON MEAT PRODUCTS Group 10 Benzoic acid Specific action against COO compounds: Aromatic Salycilic acid, gallic acid, toluic acid, phthalic the aerobic sporadic carboxylic acids acid, isophthalic acid, terephthalic acid germ of the genus bacillus which contaminates meat products Group 11: Dihydroxybenzole Aromatic polyvalent Guayacol hydroxy compounds Group 12: Acetol Desirable Hydroxy -oxo- compounds: Reaction with meat Aliphatic h ydroxyaldehydes and proteins to develop the hydroxy ketones. characteristic color of smoke Group 13: Salicylaldehyde (2-hydroxybenzaldehyde), Desirable: intense aroma Hydroxy -oxo- compounds: 4-Anisaldehyde, Vanillin and and flavor of vanilla and Aromatic compounds, phenol coniferaldehyde conifers aldehydes and phenol ketones Group 14: Glyoxal Casing hardening Compounds with various oxo - Diacetyl Margarine and bread groups: color and smell Di aldehydes and Di ketones Group 15 Maleic acid Desirable for color: Compounds with 2 or more pigment carboxyl groups: Saturated and unsaturated dicarboxylic acids Group 16 Pyruvic acid, levulinic acid Oxo - carboxy compounds : Keto acids Group 17 Pyrrole, pyrazine, indole, carbazole Darkening of color Nitrogenous organic compounds

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On the other hand, some compounds have non-desirable effects on products, such as phenol (bad taste), acetone and unsaturated long-chain compounds (undesirable aroma). Among non-desirable compounds, polycyclic aromatic hydrocarbons (PAH) are the most important. The most significant endpoint of PAH toxicity is cancer (ATSDR, 1995; ATSDR, 2009). The major contributors to PAH intake are cereals and cereal products (owing to their high levels of consumption in diets) and vegetable fats and oils (due to higher concentrations of PAH in this food group) (CAC, 2009). PAH are found in many kinds of foodstuffs in the human diet, such as smoked fish, mussels, coffee, bread and smoked cheese (Martorell et al., 2010). However, particular attention has been paid to smoked meat products because the highest levels of total PAH have been detected in these products, especially in infrequent cases of a high consumption of these products in the diet (CAC, 2009; Gomaa, Gray, Rabie, Lopez-Bote, & Booren, 1993; Karl & Leinemann, 1996; Larsson, Pyysalo, & Sauri, 1988; Martorell et al., 2010).

3.2 PAH formation and transport mechanisms

The chemical formation of PAH during meat product smoking is defined by several food technology researchers as the incomplete combustion or thermal decomposition (pyrolysis) of wood (Conde, Ayala, Afonso, & González, 2005; Djinovic, Popovic, & Jira, 2008; Gomes, Santos, Almeida, Elias, & Roseiro, 2013; Hitzel, Pöhlmann, Schwägele, Speer, & Jira, 2013; Pöhlmann et al., 2013b; Lorenzo et al., 2010; Rey-Salgueiro, García-Falcón, Martínez-Carballo, González- Barreiro, & Simal- Gándara, 2008; Škaljac et al., 2014; Wretling, Eriksson, Eskhult, & Larsson, 2010). However, for an in-depth understanding of this process, scientific research papers on PAH formation during biomass combustion should be taken into account. PAH can be found as Tertiary Tar Products formed during biomass pyrolysis (Basu, 2010). Pyrolysis products can be classified as solids (mostly char or carbon), liquids (tars, heavier hydrocarbons and water) and gases (CO 2, H 2O, CO, C 2H2, C 2H4, C 2H6, C 6H6, etc.). Tar, also known as bio-oil or biocrude, is a black, tarry fluid formed by a mixture of complex hydrocarbons containing large amounts of oxygen and water (Basu, 2010). Tar is a complex microemulsion mixture of condensable hydrocarbons, including oxygen-containing, 1- to 5-ring aromatic and complex polyaromatic hydrocarbons, among others (Devi, Ptasinski, & Janssen, 2003). The International Energy Agency (IEA) Bioenergy Agreement, the U.S. Department of Energy (DOE) and the DGXVII of the European Commission agreed to classify all components of product gas with a molecular weight higher than benzene as

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tar (Knoef, 2005). Figure 1 shows the formation of tertiary tar products as a function of the temperature of biomass conversion (Evans & Milne, 1997). Tertiary tar products are produced after the destruction of primary tar products, at about 750°C, with increasing temperature. Condensed tertiary aromatics thus make up a series of polynuclear aromatic hydrocarbons (PAH) without substituents (atoms or a group of atoms substituted by hydrogen in the parent hydrocarbon chain) (Basu, 2010; Evans & Milne, 1997). The process of producing aerosols containing PAH from the decomposition of primary tar products of biomass has been studied by several authors (Li et al., 2009; Liu et al., 2013; McGrath, Sharma, & Hajaligol, 2001; Simoneit, 2002), who found that PAH formation increases with temperature and residence time. It was also been found to depend on the cell wall components of the biomass (hemicellulose, cellulose and lignin) and may even contain heavy metals (Wobst, Wichmann, & Bahadir, 2003).

0,8

0,6

0,4

0,2

Tar conversion fraction) (weight Tar 0 500 550 600 650 700 750 800 850 900 950 1000

T (ºC) primary secondary tertiary-alkyl Condesed Tertiary aromatics: PAH tertiary-PNA

Figure 1. Temperature of polycyclic aromatic hydrocarbons (PAH) formation: tertiary tar products production during biomass conversion. (Adapted from Evans & Milne, (1997), page 804 and Basu, (2010), page 102).

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During meat processing, tar is transported through the smoking chamber by aerosols, conveying PAH to the meat products being smoked, which eventually become contaminated. Several processes occur at the same time in the traditional conversion of biomass during meat smoking. The wood starts to dry, pyrolysis commences in some parts of the fire, while complete combustion occurs in the parts nearest the flames. However, PAH contamination of meat products can have different origins. Fir st, raw ingredients can be PAH “pre -contaminated” due to atmospheric pollution. Animals (pigs, cows, etc.) and vegetables (onions, garlic, etc.) can be contaminated by PAH present in the air (by deposition), soil (by transfer) or water (deposition and transfer). There are numerous natural and mostly anthropogenic sources of PAH in the environment, including exhaust gases from motive sources (motor vehicles and aircrafts), industrial plants, forest fires, volcanic eruptions, domestic heating with open fireplaces, etc. (CX/FAC 05/37/1, 2004; CAC, 2009). Second, food technology processes other than smoking, such as packaging, drying, grilling, roasting, (Chung et al., 2011), baking, barbecuing (Kazerouni, Sinha, Hsu, Greenberg, & Rothman, 2001) and frying, can also contribute to the final PAH content of meat products (CAC, 2009). Third, some of the products in a smoking chamber are hung above others on different bars on the same shelving (see a graphical representation of the smoking room in Figure 2 or in Gomes et al. (2013)). Products are heated inside the chamber, causing their fat content to flow outward through their natural casing (Ledesma, Rendueles, & Díaz, 2015b). On the one hand, some fat falls down onto or comes into contact with the fire, thereby increasing the PAH content of the smoke (Chung et al., 2011; Janoszka, Warzecha, Blaszczyk, & Bodzek, 2004). On the other hand, some fat already contaminated with tar falls onto other products, increasing the PAH content of the latter (CAC, 2009; Viegas, Novo, Pinto, Pinho, & Ferreira, 2012). Finally, meat products are contaminated with PAH during direct smoking due to the deposition of tar aerosols from the smoke (Ledesma et al., 2015b).

4. Products and regulations

4.1 Analytical methods for PAH determination

The determination of PAH in meat products involves two critical steps: sample preparation and analytical determination. The determination of PAH in meat products depends on their fat content, which could make their pre-treatment more difficult and lengthier than that of other environmental or non-fatty food samples, such as water or beverages. Classical pre-

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treatment and analytical methods have thus been enhanced over time to determine PAH in meat. Several articles focusing on the comparison of classical and modern methods for PAH determination in foods have been published (Moreda, Pérez-Camino, & Cert, 2001; Moret & Conte, 2000; Plaza- Bolaños, Garrido Frenich, & Martínez Vidal, 2010; Šimko, 2002), the most recent to date being the paper by Purcaro, Moret, and Conte (2013). Classical pre-treatment methods included saponification, liquid –liquid extraction and Soxhlet extraction in combination with gel permeation chromatography (GPC) (Chiu, Lin, & Chen, 1997; Grimmer & Böhnke, 1975; Fretheim, 1976; Šimko, 200 2). Modern methods include microwave-assisted extraction (MAE), sonication (Ledesma et al., 2014; Purcaro, Moret, & Conte, 2009), accelerated solvent extraction (ASE) (Djinovic et al., 2008; Martorell et al., 2010; Sun, Ge, Lv, & Wang, 2012), ultrasound- assisted solvent extraction (USAE), ultrasound-assisted emulsification –microextraction (USAEME) (Yebra-Pimentel, Martínez-Carballo, Regueiro, & Simal-Gándara, 2013) and pressurized liquid extraction (PLE) (Pöhlmann et al., 2013a, 2013b), followed by SPE (García-Falcón & Simal- Gándara, 2005; Hitzel et al., 2013; Purcaro et al., 2009), stir-bar sorptive extraction (SBSE) or solid phase microextraction (SPME). SPME coupled to a direct extraction device (DED) has also been proposed (Martin & Ruiz, 2007). The combination of sonication with the SPE pre-treatment method has been found to be a good method. It entails a low level of solvent consumption and waste generation, short operating times and is easy to set up and has been found to be better than the classical combination of Soxhlet extraction and gel permeation chromatography (GPC) (Ledesma et al., 2014; Purcaro et al., 2009).

Analytical methods for identifying PAH include HPLC combined with fluorescence (FLD) or ultraviolet (UV) detectors (Lorenzo et al., 2011; Moret & Conte, 2002; Purcaro et al., 2009; Roseiro, Gomes, Patarata, & Santos, 2012; Santos, Gomes, & Roseiro, 2011) and GC combined with flame ionization (FID), ion trap (ITD) and mass spectrometry (MSD) detectors (Hitzel et al., 2013; Olatunji, Fatoki, Opeolu, & Ximba, 2014). GC-MS and GC/HRMS are widely used today to determine PAH in meat products (Djinovic et al., 2008; Hitzel et al., 2013; Martin & Ruiz, 2007; Martorell et al., 2010; Šimko, 2002; Stumpe -V!ksna, Bartkevics, Kukare, & Morozovs, 2008; Wretling et al., 2010).

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4.2 Regulation of PAH in meat products

To protect consumers against PAH intake from diet, the European Commission (EC) has adopted several regulations over the last 10 years. In accordance with the European Scientific Committee on Food (SCF) (SCF, 2002), PAH levels in foods have been regulated via EC Regulation No 208/2005 (EC, No 208/2005 ), and European Union (EU) Commission Regulation No 1881/2006 (EU, No 835/2011). Benzo(a)pyrene (BaP) was set as the marker for the occurrence and effect of carcinogenic PAH in food, also including benz(a)anthracene, benzo(b)fluoranthene, benzo(j)fluoranthene, benzo(k)fluoranthene, benzo(g,h,i)perylene, chrysene, cyclopenta(c,d)pyrene, dibenz(a,h)anthracene, dibenzo(a,e)pyrene, dibenzo(a,h)-pyrene, dibenzo(a,i)pyrene, dibenzo(a,l)pyrene, indeno(1,2,3-cd)pyrene and 5-methylchrysene. In addition, the JECFA also recommended including benzo(c)fluorene (WHO, 2006). Table 2 shows the names, abbreviations, relative molecular weights and chemical structures of the 16 PAH regulated in meat products by the European Union. A maxi mum level of 5 •g/kg BaP was set for smoked meats and smoked meat products in this EC regulation. States were asked to make further analyses of the relative proportions of these PAH in foods to inform a future review of the suitability of maintaining BaP as a marker. EC Directives 2005/10/EC (EC, No 2005/10 ) and No 333/2007 (EC, No 333/2007) established the sampling methods and the methods of analysis for the official control of the levels of BaP in foodstuffs. In accordance with a new study of data and opinion of the EFSA Scientific Panel on Contaminants in the Food Chain (CONTAM Panel) (EFSA, 2008), European Union (EU) Regulation No 835/2011 (EU, No 835/2011) (Table 3) specified that two dates must be considered as regards PAH contamination in foodstuffs. New maximum levels for the sum of four substances (PAH4) (BaP, benzo(a)anthracene, benzo(b)fluoranthene and chrysene) were introduced, maintaining a separate maximum level for BaP to ensure comparability between previous and future data. The maximum permissible BaP content in smoked meat and smoked meat products was reduced to 2.00 •g/kg wet weight as of 1/9/2014. The permissible sum of PAH4 in these foods was set at 30.0 •g/kg wet weight from 1/9/2012 to 31/8/2014 and was reduced to 12.0 •g/kg wet weight as of 1/9/2014. This regulation specified that the separate maximum level for BaP should be re-assessed based on new data that will be generated in the future. A recent study by Lorenzo et al. (2011) reports that BaP is a good marker for the sum of 15 PAH as well as for 7 PAH classified as probable human carcinogenics by the USEPA in ‘chorizo gallego’, a typical Spanish smoked meat product. A new revision of the legislation has not yet been carried out.

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Table 2. Name, abbreviations, relative molecular weights and chemical structures of the 16 polycyclic aromatic hydrocarbons (PAH) regulated in meat products by the European Union.

PAH compound Abbreviation Molecular weight Chemical structure

Benzo(a)pyrene BaP 252

Benz(a)anthracene BaA 228

Benzo(b)fluoranthene BbF 252

Benzo(j)fluoranthene BjF 252

Benzo(k)fluoranthene BkF 252

Benzo(g,h,i)perylene BghiP 276

Chrysene Ch 228

Cyclopenta(c,d)pyrene CPP 226

Dibenz(a,h)anthracene DBahA 278

Dibenzo(a,e)pyrene DBaeP 302

Dibenzo(a,h)pyrene DBahP 302

Dibenzo(a,i)pyrene DBaiP 302

Dibenzo(a,l)pyrene DBalP 302

Indeno(1,2,3cd)pyrene IP 276

5-methylchrysene 5MeCh 242

Benzo(c)fluorene BcF 216

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Table 3. New European limit for polycyclic aromatic hydrocarbons (PAH) in meat products set by EU Regulation No. 835/2011, applied since 1/9/2014.

Meat Product PAH limit (•g kg -1 ) BaP PAH4 * Smoked meat and smoked meat 2.0 12.0 products (*)PAH4: •PAH: BaP, BaA, BbF and CHR.

The USFDA regulates contaminant levels in foodstuffs, but has still not established standards governing the PAH content of foodstuffs. The maximum contaminant level goal for BaP in drinking water is 0.2 parts per billion (ppb). In 1980, the US EPA established ambient water quality criteria to protect human health from the carcinogenic effects of PAH exposure. The recommendation was a goal of zero (non-detectable level for carcinogenic PAH in ambient water). As a regulatory agency, the USEPA establishes a maximum contaminant level (MCL) for BaP, the most carcinogenic PAH, at 0.2 ppb (ATSDR, 2009; EPA, 2013).

4.3 PAH in smoked meat products around the world

Table 4 summarizes the recent PAH levels found in a number of different smoked meat products around the world studied by different researchers. The maximum BaP content found in various studies falls below the maximum value currently allowed by European Union regulations (2 •g/kg), e.g. in commercial samples from various local supermarkets in the Republic of Korea (Chung et al., 2011), some Spanish commercial meat products and chorizos (Ledesma et al., 2015a; Martorell et al., 2010), smoked meat products from Italian markets (Purcaro et al., 2009) and Portuguese traditional smoked meat products (Santos et al., 2011).

However, a high BaP content is still found in smoked meat products around the world: 36.9 •g/kg in Swedish ham produced by direct “sauna ” smoking with birch logs (Wretling et al., 2010), 10.02 •g/kg in smoked pork from Cape Town, South Africa (Olatunji et al., 2014), 6.98±2.01 •g/kg in Beef satay from a Malaysian market (Jahurul et al., 2013), 31.20 •g/kg in commercial smoked meat from Estonia (Reinik et al., 2007), 17.63 •g/kg in smoked belly of pork from Germany (Jira, Ziegenhals, & Speer, 2008), and 3.21 ± 0.12 •g/kg in commercial smoked chorizos from Spain (Ledesma et al., 2015a). It is thus still important to control and study manufacturing conditions in order to minimize PAH contamination of smoked meat products.

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Table 4. Polycyclic aromatic hydrocarbons (PAH) in different smoked meat products worldwide.

References Studied variable Sample Studied smoking PAH results interval (•g/kg) BaP (•g/kg) PAH (•g/kg) conditions Maximun Minimun Maximun Minimun Chung et al., Commercial samples from Ham, bacon, processed Commercial samples 0.08 0.01 PAH 2 PAH 2 (2011) various local supermarkets in the products and sausages from various local Sausages Processed products 1.09 0.15 Republic of Korea. (smoked products). supermarkets in the Bacon Processed Republic of Korea. products Jahurul et al., Different kinds of chicken, beef, Different kinds of chicken, Grilling, boiling, frying, 6.98±2.01 n.d in any products (2013) mutton, sausage and nuggets beef, mutton, sausage and stir frying, Beef satay except for : from a Malaysian market. nugget s from a Malaysian (grilled) - Chicken satay ( grilled) market. -Sausage (fried) -Beef satay (grilled) Jira et al., 22 smoked meat products, Smoked raw meat products Commercial hams from 17.63 0.02 BaP can be used as the leading (2008) mainly very sm oked hams (mainly raw smoked hams) different producers. Smoked belly of Smoked ham substance in smoked meat from different producers: pork with a products. This has to be confirmed Ham, belly pork ham and strongly fading by the investigation of smoked wild boar ham black surface. representative samples. Ledesma et al., 16 smoked meat products 16 chorizos asturianos Commercial chorizos 3.21 ± 0.12 0.38±0.08 (201 5a) (chorizos) from different without casing. from different producers. producers in Spain. Lorenzo et al., Spanish traditional smoked 16 Chorizos gallegos without Chorizos from 16 PAH 1 PAH 1 (2011) sausage varieties: “Chorizo casing. different industrial pork 6.76 5.01 gallego” and “Chorizo de 16 Chorizos de cebolla manufacturers following Chorizo de Chorizo gallego cebolla”. without casing. traditional methods. cebolla (16 different manufacturers) Martorell et Food and intake by the Veal steak, hamburger, loin 1.10 <0.06 Total PAH 3 Total PAH 3 al., (2010) populatio n of Catalonia, Spain. of pork, pork sausage, Salami Veal steak, l oin of 364.91 1.18 Loin of pork chicken breast, lamb, boiled pork Salami ham, Frankfurt sausage, salami, cured ham.

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Table 4. (continued). Olatunji et al., Processed meat products from Beef stripe fillet. Smoking 10.02 n.d (2014) Cape Town, South Africa. Pork fillet. Grilling smoked pork Unprocessed pork Chicken fillet. Boiling and chicken Purcaro et al., Smoked meat products from an Smoked bacon, smoked “Pitina”: Smoking in a 0.8 <0.05 (2009) Italian market speck, smoked pork meat, WUDGLWLRQDOZD\SODFLQJWKH Pitina smoked bacon, smoked smoked beef meat, pitina. pork and beef meats PHDWQHDUWKHILUHSODFHIRU VHYHUDOGD\V Reinik et al., Commercial meat products in Smoked sausage, ham, meat Commercial meat 31.20 <0.3 in all products PAH 4 PAH 4 (2007) Estonia 2001 –2005. and chicken. products Smoked meat 8.0 5.7 Smoked ham smoked chicken Roseiro et al., Portuguese traditional meat and Samples, (%fat): Combustion of wood M: 4.75 M:0.36 PAH4 PAH4 (2012) blood sausages A) “Alentejo’’ products: from: i) Salp icão M: 294.50 M: 3.47 Smoked meat (M): “Alentejo ” from c) Paio tradicion. i) Salpicão c) Paio tradicion. a) Chouriço de carne (25.1%), Quercus ilex and/or “Trás-os - from “Alentejo” from from b) Paínho (24.2%) and c) Paio Quercus suber wood. Montes ” “Trás-os- “Alentejo” tradicional (40%). Smoking times (days): Montes ” Blood sausages (BP): a) 5, b) 15, c) 30, d) 8, e) d) Chouriço mouro (53%), e) 6, f) 8. B.P:0.32 Cacholeira (41.8%) and f) BP: 5.65 f) Morcela Morcela (46.4%). ‘‘Trás -os- Montes’’: l) Moura from “Alentejo” B.P: 271.83 B.P: 1.84 Quercus faginea and/or from B) “Trás-os- Montes ” Castanea sativa Miller “Trás-os - l) Moura f) Morcela from products: wood. Montes ” of “Alentejo” Smoked meat (M): ‘Trás -os- g) Alheira (16%), h) Chouriço Smoking times (days): Montes’’ de carne (20.6%) and i) g),h),i),j),k),l): unknown Salpicão (14.4%) Blood sausages (BP): j) Chouriço doce (11.7%), k) Morcela (39.5%) and l) Moura (23.9%). Natural casings.

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Table 4. (continued). Santos et al., Portuguese traditional smoked Samples, (%fat): Direct traditional Mean contents (2011) meat products MEAT SAUSAGES (M): smoking M: 0.63 M:0.36 PAH4 PAH4 a) Chouriço de carne (25.1%), Wood: Quercus ilex . M: 4.80 M: 3.47 b) Paínho (24.2%) and c) Paio Smoking time (days): b) Paínho c) Paio tradicion. b) Paínho c) Paio tradicional tradicional (40.0%) a) 5: b, 15; c) 30; d) 8; e)6 BLOOD SAUSAGES (BS): ; f) 8. B.P:0.32 B.P: 1.84 d) Chouriço mouro (53.0%), BP: 0.39 f) Morcela B.P: 6.94 Morcela e) Cacholeira (41.8) and f) e) Cacholeira d) Chouriço Morcela (46.4). mouro All samples stuffed in natural BaP max and min: 1.00 (painho), 0.21 (morcella) casing.

Wretling et al., Swedish smoked meat Ham, bacon, pork tenderloin, Different producers with 36.9 n.d PAH4:209 PAH4: n.d (2010) pork shoulder, elk, heart, elk the following smoking Ham smoked Ham, ham indirect and reindeer meat combined, systems: by direct alder chips. Ham smoked Ham, pork lamb, leg, reindeer meat, a) Direct, b)I ndirect, sauna with Pork shoulder, lean by direct sauna tenderloin and lean, chicken, turkey, c)“ Direct sauna”. birch logs reindee r meat, chicken, with Birch shoulder, lean turkey, sausages . sausages, elk sausage. Fuels: Sallow, alder or logs reindeer meat, birch logs. Sallow or alder chicken, turkey, chips. sausage, elk sausage.

PAH4: !PAH: Sum of BaP, BaA, BbF and CHR. 1: !PAH: US EPA PAH7: BaP, BaA, CHR, BbF, BkF, DhA, IcP. 2: !PAH: PAH: CHR, BbF, BkF, BaP, DBahA, BghiP, IP 3: !PAH: Na, Ap, Ac, F, Pa, A, Fl, P, BaA, Ch, BbF, BkF, BaP, IP, DBahA, BghiP. 4: !PAH: BaA, BbF, BjF, BkF, BghiP, BaP, Ch, DBahA, DBaeP, DBalP, IP, 5MeCh. n.d: non-detected.

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5. Prevention of PAH contamination of smoked meat products

The Codex Alimentarius Commission (CAC) established by FAO and WHO recognizes the benefits of smoking, including the availability of food products produced by traditional smoking and the prevention of deterioration, microbiological contamination and growth. The “Code of practice for the reduction of contamination of food with (PAH) from smoking and direct drying processes” (CAC/RCP 68/2009) stipulates that PAH contamination in foods tuffs via food processing should be minimized, providing producers with the 10 variables that need to be controlled. Figure 2 shows a scheme of these variables in a chamber during direct smoking of meat products. These include the kind of fuel (different woods and other plant materials, diesel, gases, liquid/solid waste and other fuels), smoking or drying method (direct or indirect), the process of generating smoke in relation to the temperature of pyrolysis and airflow in the case of a smoke generator (friction, smoldering, thermostated plates) or in relation to other methods, such as direct smoking or regenerated smoke by atomizing smoke condensate (liquid smoke), the distance between the food and the heat source, the position of the food in relation to the heat source, the fat content of the food and its evolution during processing, the duration of smoking and direct drying, the temperature during smoking and direct drying, the cleanliness and maintenance of equipment, and the design of the smoking chamber and the equipment used for the smoke/air mixture.

All these variables should be controlled in each smoking process. For this reason, the difficulty in controlling the PAH content of smoked meat products has been highlighted by several researchers (Djinovic et al., 2008, Lorenzo et al., 2011; Roseiro et al., 2012; Santos et al., 2011; Stumpe- V!ksna et al. , 2008). Table 5 summarizes the latest published studies on the parameters affecting the smoking process with the aim of understanding PAH contamination of smoked meat products. An overview of these studies is presented next.

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Figure 2. Smoking chamber: Representation of CAC/RCP 68/2009 variables to control polycyclic aromatic hydrocarbons (PAH) contamination of meat products in direct and indirect smoking processes.

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Table 5. Codex alimentarius (CAC/RCP 68/2009) parameters studied by researchers with the aim of controlling PAH contamination (•g/kg) of smoked meat products. CODEX References Studied variable Sample Studied smoking conditions PAH re sults interval (•g/kg) ALIMENTARIUS BaP (•g/kg) PAH (•g/kg) PARAMETER Maximun Minimun Maximun Minimun Hitzel et Wood: Beech ( Fagus Samples: (predried). Sheep Equipment: T1900 smoking F: F: F: F: al., (2013) sylvatica ), oak ( Quer cus casings, sub -mucosa of small chamber, Alder BA Alder BA robur ), spruce ( Picea intestine. smoke generator (RZ 325) 0.80±0.15 0.32±0.05 (4.70±0.49) (2.16±0.31) abies ), poplar ( Populus -Frankfurter -type sausages (Fessmann). spp. ) and fir ( Abies alba ) (F) Pretr eatment of samples: M: M: M: M: from Rettenmaier & 29.6% fresh pork, 19.8% -F: reddening for 10 min at Alder Oak Alder Poplar Söhne (Rosenberg, fresh beef, 25.9% back fat, 52 °C and drying for 12 min at 56° C. 0.60±0.01 0.23±0.03 4.38±0.15 (2.37±0.11) a) Fuel: Germany) and alder 22.6% ice, 1.4% salt -M: drying and curing for 2 days at BC BC woods and other (Alnus spp. ) and hickory (containing sodium nitrite 22 °C in a climatic chamber. 0.60±0.29 5.00±1.51 plant materials, (Carya spp. ) from 0.4%), 0.04% ascorbic acid, Smoking conditions diesel, gases, Thomsen GmbH & Co. KG 0.17% dipotassium .Smoking time: 12 min (F), 30 min (PAH4) (PAH4) liquid/solid waste (Handewitt, Germany). hydrogen phosphate, and (M). and other fuels. Spiced beech wood chips 0.42% “Goldwürstchen” -Smoking temperature: 58 °C (F), with apple smoking spice spice mix. 22 °C (M) mix (BA), spiced beech -Mini -salamis (M) -Smoke de nsity: intensive. wood chips with cherry 76.9% frozen pork, 19.6% -Ventilator velocity: 3000 rpm smoking spice mix (BC), frozen Post treatment of samples: spiced beech wood chips back fat, 2.4% salt F: Scald ing for 25 min at 75°C. with juniper berries and (containing 0.4% sodium bay leaves, fir. nitrite), 0.4% glucose, 0.4% spice mix “Mild French- Style Salami ” from Raps and at least 1011 “Optistart Sprint ” active microorganisms from Raps. Stumpe- Wood: Apple, alder, Pork meat Smoking: home kiln Aspen Apple (PAH 1) (PAH 1) V!ksna et alder + juniper, spruce, Smoking temperature: 80 °C 35.07 6.04 Spruce Apple al., (2008) maple, hazel, plum, Smoking tim e: 5 hours 470.91 (47.94) aspen, bird -cherry, rowan tree, charcoal.

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Capítulo 4 Table 5. (continued). CODEX References Studied variable Sample Studied smoking conditions PAH results interval (•g/kg) ALIMENTARIUS BaP (•g/kg) PAH (•g/kg) PARAMETER Maximun Minimun Maximun Minimun b) Smoking or Gomes et Direct (d) and indirect Lean and fatty pork trimmings, 4hours/day, 8 days, combined with 0.32 0.09 PAH4:10.35 PAH4: 4.21 drying method (i) smoking placing an salt (1%), garlic paste (3%), drying in a controlled environmental al., (2013) (direct or obstacle (a stainless paprika paste (3.5%), curing chamber (5 –15°C; 25 –55% relative Hog casing, Collagen Hog casing, Collagen indirect). steel plate ) above the salts [0.25% (NaNO 3 4.9%; KNO 3 humidity).Oak wood ( Quercus ilex L .), casi ng, casing, smoke generator, or 5%), antioxidants [0.15% 2 smoking expos ures: Direct (d) and a:20% fat, not . (sugars; E301 relative indirect (i) , placing an obstacle (a indirect a:20% fat, b:40% fat, a:20% fat, composition unknown) and stainless steel plate ) above the smoke smoking indirect indirect direct smoke water(2.5%)].Fat(a)20% ,(b): 40%. generator , or not. smoking smoking c) Smoke Pöhlmann Different conditions of Frankfurter-type sausages: 1.8 Equipment: FPC 100 smoking chamber 0.48±0.09 0.11±0.04 2.96±0.43 1.10±0.22 generation et al., glow smoke kg fresh pork, 1.2 kg fresh beef, from Fes smann. process in (2012) 1.62 kg back fat, 1. 38 kg ice, Pretreatment of samples : T:58 °C T: 58 °C T:58 °C T: 58 °C relation to the 84.5 g salt (containing NaNO 2); Reddening for 10 min at 52°C and t:11 min t:28min t:11 min t:28min temperature of 0.4%, 2.5 g ascorbic acid, 10 g drying for 12 min at 56°C. SD: i SD: l SD: i SD: l pyrolysis and to dipotassium hydrogen Smoking times (min) (t): 10,11,12, V: V:750rpm V: 1500rpm V:750rpm airflow in the phosphate (K2HPO4) and 26 g 13,14,20,21,22,28,29,30. 1500rpm M:12,2% M:12,6% M:12,2% case of a smoke “Goldwürstchen” spice mix from Temperature (T): 58°C. Smoke density M:12,6% (PAH4) (PAH4) generator(frictio Raps. Sheep casing from (SD): intensive(i), medium(m), light(l). Comparison of samples of similar moisture n, smoldering, submucosa of the small Ventilator velocity (V) (rpm): 750, thermostated intestine. 1500, 3000.Moisture content of wood plates) or with (M)(%): [9.8-29.4] other methods Pöhlmann Smoldering (S), Steam Frankfurter-type sausages: S: BaP Means PAH4 means such as direct et al., (St), Friction (F), Touch 29.5% fresh pork, 19.6% fresh 10,20,28 min.(i,m,l).750rpm (a). S: 0.27 ± 0.14 S: 1.88±0.69 smoking or liquid (2013a) smoke (T) at different beef, 26.4% back fat, 22.5% ice, 11,21,29 min. (i,m,l).1500 rpm (b). (l):0.16 (l):1.32 smoke. smoking times, 1.4% salt (containing sodium 12,22,30 min. (i,m,l).3000 rpm (c). (m):0 .24 (m):1.71 ventilator velocities nitrite 0.4%), 0.04% ascorbic St: (i):0.40 (i):2.60 [rpm]: 750rpm (a), acid, 0.17% dipotassium 3, 4, 5, 6, 8 min at 750 rpm (1 study 4 (a): 0.18 ± 0.07 (a): 1.51 ± 0.39 1500rpm (b) and hydrogen phosphate and 0.42% min with 3000 rpm), Different steam (b): 0.29 ± 0.16 (b): 1.96 ± 0.83 3000rpm (c) and smoke “Goldwürstchen” spice mix smoke temperatures. (c): 0.34 ± 0.15 (c): 2.17 ± 0.72 densities: intensive (i), from Raps. Sheep casings made F: ventilator velocity (1500 rpm) St: 0.08 ± 0.07 St: 0.94± 0.66 medium (m), light (l). from the submucosa of the 26, 37 and 40 min, 1.2,2 and 2.8 bar, F: 0.03 ± 0.01, F: 0.29 ± 0.11 small intestine. 30, 60 and 240sec interval of friction. T: 0.09 ± 0.02 T: 1.57 ± 0.32 T:15,20,25,30,35,40 min at 750 rpm 200

Resultados Table 5. (continued). CODEX References Studied variable Sample Studied smoking conditions PAH r esults interval (•g/kg) ALIMENTARIU BaP (•g/kg) PAH (•g/kg) S PARAMETER Maximun Minimun Maximun Minimun d) Position of Pöhlmann et Different positions in the Sheep casing. Positions in the smoking 1.10 0.88 PAH4 PAH4 the food Fresh pork, Fresh beef , Back fat, chamber: centre back back 4.57 3.62 al., (2013b) smoking chamber with respect ice : Front (a), cent er- front (b), centre back back to the heat 29.4%, 19.6%, 26.5%, 22.5%. cent er-back (c), back (d). source. T1900 smoking chamber, Fessmann. Smoking time:12 min Smoke density: intensive. Ventilator velocity: 3000 rpm f) Fat Pöhlmann et Fat content (F): Sheep casing. T1900 smoking chamber, 1.37 0.28 6.2 1.5 content of al., (2013b) 10%,20%,30%,39% Fat (F), Fresh pork, Fresh beef , Fessmann. (PAH4) (PAH4) the food and Back fat, ice : Smoking time : F:39.1% F:9.9% F:19.6% F:9.9% what 10%, 35.8%, 23.9%, 9.9%, 28.3% (t):12,13,14,15 min t: 15min t: 12min t: 15min t: 12min happens to it 20%, 32.0% ,21.3%, 19.6%, 25.0% Smoke density (SD): SD: i SD: m SD: i SD: m during 30%, 28.0%, 18.7%, 29.5%, 21.8% intensive (i) and medium V: 3000 V: 1500 V:3000 V:1500 processing. 39% 24.1%, 16.1%, 39.1%, 18.7 % (m). Ventilator vel ocity (V) (rpm): 750, 1500, 3000. g) Duration Djinovic et Smoking time: Beef ham (a) Beech wood combustion. BaP, days BaP, days PAH 2, days PAH 2, days of smoking a,e: 0,6,9,12,15,18 days Pork ham (b) Distance from the smoking a:1.09, 18 a: 0.02, 0 a: 21.3, 18 a: 0.4, 0 al., (2008) and direct b,c,d,f: 0,6,9,12,15 days Bacon without skin (c) source: b:0.52, 15 b: 0.04, 0 b: 10.2, 15 b: 0.9, 0 drying. Bacon with skin (d) a,b,c,d: 2m c:1.04, 15 c: 0.03, 0 c: 22.7, 15 c: 0.6, 0 Cajna sausage (e) e,f: 5 m. d:0.55, 15 d: 0.03, 0 d: 12.2, 15 d: 0.7, 0 Sremska sausage (f) e:0.24, 18 e: 0.18, 0 e: 4.4, 18 e: 3.5, 0 f: 0.33, 15 f: 0.08, 0 f: 10.5, 15 f: 2.0, 0 Ledesma et Smoking time: 0,1,3,5,7 days Chorizo: Length:13.7 cm, mass: Traditional direct smoking. BaP, days BaP, days al., (2014) Product depth: 115.1 g . Ingredients: Pork loin oak (90%) and chestnut 0.75±0.05,5 <0.24, 0 D1: casing (1.15g) ; D.2: 0.25 cm (46.8%), pork jowl (46.8%), salt (10%) woods D4 from the casing to the casing (1.8%), garlic (1%), sweet or spicy Distance from the smoke D1 0.88±0.10 (23.6g).D.3 Part between D2-D4 paprika (2%) and herbs (1.6%). source: 20.0±1.1, 5 days (65.4g) . D.4: 0.25 cm from the Antioxidants colorings, emulsifiers 10 m. 5 da ys center and the center (25.0g). and otheradditives.Natural casing.

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Capítulo 4 Table 5. (continued). CODEX References Studied variable Sample Studied smoking conditions PAH r esults interval (•g/kg) ALIMENTARIU BaP (•g/kg) PAH (•g/kg) S PARAMETER Maximun Minimun Maximun Minimun García- Natural casing (N) Chorizo: pork meat, water, paprika, Smoking room:3.5m X 1.6m X 2.7m- 2 1 57 22.1 Falcon & Collagen casing (C) salt and oregano. Wood: Q . robur Natural Collagen •PAH 3 •PAH 3 Simal - Fat: Distance to the smoking source: 2m. cased cased Natural Collagen Gándara, a) 46% (low fat) Smoking time: 8 days. chorizo with chorizo with casing cased casing with (2005) b) 53% (high fat) Drying after smoking: 16 days. 53% fat 53% fat,both chorizos , low low fat Natural (N) and collagen (C) casings. chorizos fat content content with 46% fat (46%) (46%) Gomes et al., Collagen casing (C) Lean and fatty pork trimmings, salt 4hours/day, 8 days, combined with 0.32 0.09 PAH4:10.35 PAH4: 4.21 Hog casing (H) (1%), garlic paste (3%), paprika drying in a controlled environmental Hog (natural) Collagen Hog casing, Collagen (2013) paste (3.5%), curing salts [0.25% chamber (5 –15°C; 25 –55% relative casing, casing, casing, (NaNO 4.9%; KNO 5%), humidity). 3 3 antioxidants [0.15% (sugars; E301 Oak wood ( Quercus ilex L .), a:20% fat, a:20% fat, b:40% fat, a:20% fat, relative composition unknown) and 2 smoking expositions: indirect indirect direct indirect k) Casing water (2.5%)]. Fat content: Direct (d) and indirect (i) placing an smoking smoking smoking smoking (a) 20% , (b): 40%. obstacle (a stainless steel plate above the smoke generator , or not.)

Ledesma et Natural casing (N) Chorizo ingredients: Traditional direct smoking. n.d 1.71 ± 0.15 al., Collagen casing (C) Pork loin (46.8%), pork jowl (46.8%), oak (90%) and chestnut (10%) (2015b) salt (1.8%), garlic (1%), sweet or woods unsmoked Chorizo in spicy paprika (2%) and herbs (1.6%). smoking time: 7 days. chorizo, and natural Antioxidants (sodium citrate or Distance from the smoke source: chorizo in casing. sodium ascorbate ), colorings 10 m. synthetic 19.9 ± 2.9 (carmine ) emulsifiers ( sodium casing Casing only triphosphate ), food preservatives (collagen) (sodium nitrite ) and other additives. Natu ral and collagen casings

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Resultados Table 5. (continued). CODEX References Studied variable Sample Studied smoking conditions PAH r esults interval (•g/kg) ALIMENTARIU BaP (•g/kg) PAH (•g/kg) S PARAMETER Maximun Minimun Maximun Minimun k) Casing Pöhlmann et Collagen Frankfurter-type sausages: T1900 smoking chamber, Fessmann. 0.81 0.08 4.77 0.58 casing(C) Fresh pork, Fresh beef , Back fat, Smoking time: 12 min. al., (2013b) Cellulose - ice : Smoking temperature: 58 °C. Sheep Peeled Sheep Cellulose peelable casing 29.4%, 19.6%, 26.5%, 22.5%. Smoke density: intensive cased Cellulose cased cased (Ce) Before smoking , samples were Ventilator veloci ty: 3000 rpm sausage cased sausage sausage Sheep casing (S) reddened for 10 min at 52 °C, and sausage then dried for 12 min at 56°C.

Škaljac et al., Natural casing -Natural (pig colon; 400-500 mm 1.Traditional conditions: n.d n.d PAH 4 PAH 4 (2014) (N) 125 long; 38 -55 mm in d iameter) Wood: sweet cherry. Collagen casing -Collagen casings (500 mm long; Distance from smoking source: 3m. 495 ± 7.65 54.1 ± 4.25 (C) 55 mm in diameter). Temp. and humidity of the smoking room: (end of (end of Traditional fermented sausage A) [8.10 to 14.6°C] (average 10.7°C), [70.1 to 91.6%] storage) storage) from Serbia “Petrovská klobása”. (av erage 78.8%), B) [8.30 to 10.7°C] (average 9.30°C) Lean pork meat (80 %) and pork 220 ± 6.30 31.3 ± 0.55 [60.7% to 90.4%] (average 76.8%). fat (20 %) home -made spicy Smoking time: 10 days. paprika powder (2.50 %), salt Drying (after smoking):90 days. (end of (end of (1.80 %), crushed garlic (0.20%), Stored conditions: 10 °C, 75%, until 270 days. drying) drying) caraway (0.20 %) and sugar (0.15 2. Industrial conditions : %). Smoke generator (indirect smoking) Natural Collagen Wood: b eech casing. casing. Moisture content of all samples: Smoke T emp.: 28.0°C. Traditional Industrial less than 35.0%. Temp. , humidity chamber: 10.3°C, 83.8% smoking smoking Smoking time: 6 hours (in 3 days). (direct) (indirect) Drying (after smoking):45 days. Storage conditions: 10°C, 75%, until 270 days. 1: !PAH: CPP, BaA, CHR, 5MC, BbF, BjF, BkF, BaP, IcdP, DBahA, BghiP, DalP, DaeP, DaiP, DahP. 2: !PAH: BcL, BaA, CPP, Ch, 5MC, BbF, BjF, BkF, BaP, BgP, DhA, IcP, DeP, DhP, DiP, DlP. 3: !PAH: F, BaA, BbF, BkF, BaP, DahA, BghiP, IcdP. 4: !PAH : ! 13 US -EPA PAH: Acy, Fln, Phe, Ant, Pyr, BaA, CHR, BbF, BkF, BaP, IcP, DhA, BgP. n.d: non-detected.

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Capítulo 4

5.1 Kind of fuel

The studies by Stumpe- V•ksna et al. (2008) and Hitzel et al. (2013) stand out for researching the influence of the kind of fuel used during meat product smoking. In both studies, the samples smoked with apple (Stumpe- V•ksna et al., 2008) and spiced beech wood chips with apple smoking spice mix (BA) and oak (Hitzel et al., 2013) were found to have the lowest BaP and PAH contents. However, the BaP contents found in the products were very different, 6.04 •g/kg (apple), 0.32 •g/kg (BA) and 0.23 (oak), respectively. This difference could be explained by the differences in smoking time, temperature and samples analyzed in each study: 5 hours, 80°C and pork meat in Stumpe- V•ksna et al. (2008) (higher BaP content); and 12 minutes, 58°C and a mix of pork and beef meats (probably less fat) in Hitzel et al. (2013) (lower BaP content). According to Hitzel et al. (2013), low BaP contents have been found in meat products smoked with oak wood; for instance, 0.75 •g/kg after 5 days smoking chorizo (Ledesma et al ., 2014) and 0.32 •g/kg after 4 hours/day over 8 days of smoking Portuguese dry fermented sausages (a product similar to chorizo, but with a lower fat content) (Gomes et al., 2013), both products being stuffed in natural casings. Products smoked with aspen, (Stumpe- V•ksna et al., 2008) and alder (Hitzel et al., 2013) have the highest BaP contents (35.07 •g/kg , 0.80 •g/kg ), respectively. Chung et al. (2011) studied the PAH content of grilled and roasted meat products, reporting a BaP content of 8.49 •g/kg in a pork shoulder loin exposed to charcoal grilling for only 30 min. However, they used gasoline on the charcoal to start the fire. Using this type of fuel greatly increases the PAH of meat products. CAC/RCP 68/2009 recommends discouraging the use of resinous woods or woods treated with chemicals (for preserving, waterproofing, fireproofing, etc.) and other fuels rather than natural woods (even as partial components) such as diesel oil, rubber (for example, tyres) or waste oil.

5.2 Smoking or drying method (direct or indirect), grilling and barbecuing.

CAC/RCP 68/2009 established that replacing direct smoking with indirect smoking (such as the use of a friction smoke generator) can significantly reduce PAH contamination of smoked foods. Gomes et al. (2013) studied the influence of direct (d) and indirect (i) smoking, placing an obstacle (a stainless steel plate) or not above the smoke generator, providing a detailed graphical scheme of the smoking room. They found a reduction in the PAH content of indirectly smoked meat products, especially those stuffed in natural (hog) casing. The reduction ranges between 33 and 45 %. CAC/RCP 68/2009 states that domestic food preparation such as roasting, baking,

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barbecuing and frying are recognized as important sources of PAH contamination. Home barbecuing and grilling could be classified as direct methods of PAH contamination. Grilled or barbequed meat was the cause of a significant proportion (21%) of the average total daily BaP intake in USA, according to the data obtained by Kazerouni et al. (2001).

Table 6 shows recent studies on PAH in grilled meat products. Various studies have been conducted on the influence during the grilling and barbecuing of meat products of marinating (Farhadian, Jinap, Faridah, & Zaidul, 2012), preheating and wrapping (Farhadian, Jinap, Hanifah, & Zaidul, 2011), the addition of onion and garlic (Janoszka, 2011), home cooking, and commercial samples from different countries, like Estonia (Reinik et al., 2007), Denmark (Aaslyng, Duedahl- Olesen, Jensen, & Meinert, 2013), Korea (Chung et al., 2011), Malaysia (Farhadian, Jinap, Abas, & Sakar, 2010) and Kuwait (Alomirah et al., 2011).

In order to reduce the PAH content of grilled meat products, the best reported pretreatment methods are steam and microwave preheating, and aluminum wrapping (100% reduction in the two carcinogenic PAH, BaP and BbFln) (Farhadian et al., 2011), an acidic marinade containing 1.2% lemon juice (70% reduction of PAH) (Farhadian et al., 2012) and the addition of onion and garlic as meat additives (Janoszka, 2011) (decrease of 60% in the total content of 6 PAH in meat samples). According to Jägerstad and Skog (2005), marinating can often result in a charred meat surface with high PAH levels. This could be caused by the type of marinade applied. Farhadian et al. (2012) recommends the following order: basic-lemon>basic> basic-oil-lemon> basic-oil. The highest BaP contents found in commercial and home-grilled samples were 24 •gkg -1 in Danish beef (Aaslyng et al., 2013), 12.5 •gkg -1 in Malaysian beef satay (Farhadian et al., 2010), 8.49 •gkg -1 in pork from Korea (Chung et al., 2011) and 5.79 •gkg -1 in meat burgers from Kuwait (Alomirah et al., 2011). BaP was not found in grilled products in several studies (Aaslyng et al., 2013; Alomirah et al., 2011; Chung et al., 2011; Farhadian et al., 2010).

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Capítulo 4 Table 6. Polycyclic aromatic hydrocarbons (PAH) in grilled meat products. Reference Studied variable Sample Studied grilling conditions Results interval PAH (•g/kg) Conclusion Maximum Minimum Aaslyng et al., Meat products Boneless pork loin Normal barbecue practice by Beef: 0 content found Beef, pork and chicken were affected (2013) barbecued at (intramuscular fat content of consumers in Roskilde 24 in all samples differently by the barbecu ing process. home in 1.4% –2.0%, 1.9% average fat (Denmark). The content of some of the PAH varied Denmark . content (3mm) sliced into 14 Chicken shows significantly between the 3 meat pieces of 2 cm -thick chops/loin. the lowest max products (pork, beef and chicken). Bee f strip loin : (intramuscular BaP content: However, time –temperature seemed fat content of 2.7% –5.9%, 4.6% to be the mo st important factor average fat content ) sliced into 1.2. compared with the type of meat. 10 pieces of 2 -cm steaks per strip of loin. Chicken breast fillets (0.6% – 1.0%, intramuscular fat content, 0.9% average fat content ). 1 muscle/sample. Alomirah et al., Grilled and 28 lamb meat (4 mandi, 6 -Charcoal grilled (indirect BaP BaP Meat tikka, whole grilled chicken, meat (2011) smoked foods kabab, 4 tikka, 6 shawerma, 3 heat): Mandi (meat and 5.79 0 content in all burger and grilled vegetables were the from Kuwait burger, 2 smoked meat and 3 chicken) Meat burger samples except major contributors to the daily intake arayes), 21 chicken (4 mandi, 4 -Charcoal grilled (direct heat): for Meat tikka, of BaP, PAH8 and SBaPeq for the shish tauk, 4 whole grilled Meat kabab, meat tikka, meat !PAH 1 smoked meat child/adolescent and adult population chicken, 6 shawerma and 3 arayes, Shish tauk, whole 129 2 Meat and Chicken in Kuwait. burger). grilled chicken. tikka mandi. -Gas grilled (indirect heat) Major influence of the type of heat Shawerma (meat and chicken). Meat tikka !PAH 1 source, duration of grilling, geometry -Electric grilled (indirect heat) contained the 6.41 of the grill a nd the use of marinating Burger (meat and chicken). highest mean chicken burger sauces and fat content. -Electric smoked (indirect concentrations heat): Smoked meat of BaP and !PAH 1 (2.48, 648), respectively

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Resultados Table 6. (continued). Reference Studied variable Sample Studied grilling conditions Results interval PAH (•g/kg) Conclusion Maximum Minimum Chung et al., Grilling and Samples (%fat) Gas roasting: 30 min at 200 °C. BEEF: nd (2011) roasting o f meat Beef: loin (18.4 -18.7%), Charcoal roasting: 30 min at 200 °C. 0.916 Charcoal grilling produces products ribs (6.7 -7.5%) and ribs Grilling: 2 kg of charcoal were placed in the bottom of Charcoal in all samples the highest PAH levels with sauces (14.0 -16.6%). the grill and 100 mL of gasoline were poured onto grilling of except for Raw pork: shoulder loin charcoal to start a fire. The beef and pork samples Ribs charcoal -grill ed (13.2 - 14.9%), belly (17.7- were grilled for 30 min. ribs and ribs 20.1%) and ribs with with sauces of sauces (14.06 -16.6%). PORK: beef and pork From local supermarkets 8.49 in the Republic of Korea. Charcoal Samples cut into small 0.5 grilling of cm cubes. shoulder loin pork. Farhadian et Malaysian 1. Charcoal grilled (well 1. Charcoal grilled (well done): BaP BaP The highest concentration al., (2010) popular grilled done): a) Ayam bakar a) 4–5 pieces of chicken, 6 –7 min grilling o n each side 12.5 n.d of PAH was detected in meat dishes (chicken), b) beef satay, c) using garden -type grill fuelled by charcoal. Beef satay in a ll samples charcoal -grilled followed chicken satay. b) 4–5 small pieces of beef, 5 –6 min grilling, on each (b) except for a) by flame -gas grilled and 2. Gas grilled – direct heat side using a satay-type grill fuelled by charcoal. ayam bakar oven grilled dishes. In (well c) 4–5 small pieces of chicken. 4 –5 min grilling on (chicken), b) charcoal grilled dishes. done):d) Beef kebab, e) each side using a satay type grill fuelled by charcoal. beef satay, and Beef satay had the highest chicken kebab, f)grilled 2. Gas grilled – direct heat (well done) c) chicken concentration of PAH. chicken. d) Sliced beef skewered onto a vertical rotisserie with satay. PAH concentrations of 3. Oven grilled – indirect a vertical gas heat element. flame -gas grilled dishes heat e) Sliced chicken skewered onto a vertical rotisserie was found to be low when (well done): g)Tandoori with a vertical gas heat element. the gas -flame source was (chicken), h) Grilled f) 1 large piece of chicken was grilled for 8 –10 min on vertical. Furthermore, chicken. each side on a gas stove. there were no significant 3. Oven grilled – indirect heat difference in PAH g) 2 pieces chicken cooked in a clay oven (Tan door. concentrations (p < 0.5) The ) heat source was gas under a clay surface. between beef & chicken h) 1 whole chicken (1 kg) cooked in an electric oven. grilled by a vertical source.

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Capítulo 4 Table 6. (continued). Reference Studied variable Sample Studied grilling conditions Results interval PAH (•g/kg) Conclusion Maximum Minimum Farhadian et Preheating and Beef (filet) and Samples of small uniformly sized pieces (about 0.5 BEEF: BEEF and The steam and microwave al., (2011) wrapping of chicken (breast cm). 3.16 ± 0.41 CHICKEN: preheating treatments and charcoal grilled and leg with skin) Marinating in satay spice: a mix of onion, sugar, aluminum wrapping have the meat products. exposed to cooking oil, lemon grass and lemon -juice for 4 h and CHICKEN: n.d same , maximum effects (100%) different kept in the refrigerator (4ºC) until use. 2.44 ± 0.26 in in reducing the two carcinogenic preheating and 1. Pretreatments: Steam and PAH (BaP and BbFln) for beef wrapping 1.1)Preheating: Both charcoal microwave and chicken meat samples. The methods. a) Microwave preheating: 40, 60 and 120 s for beef and grilled (8 and 6 preheating effect of banana wrapping on 20, 40 and 60 s for chicken samples at 180°C. min for beef and these two PAH was also quite b) Steam preheating: 100 °C for 2, 4 and 6 min for beef and chicken aluminum satisfactory for beef samples. For and 1, 2 and 3 min for chicken. respectively) wrapping chicken samples, wrapping 1.2) Wrapping samples samples. treatments (aluminum and c) Aluminum wrapping without banana) were shown to cause a d) Banana leaf wrapping preheating greater reduction in P AH 2.Charcoal grilling: treatments compared to preheating Samples skewered onto 12 satay sticks (4 pieces on treatments (steam and each stick) and processed on the satay charcoal grill). microwave). Duration: Between 2 and 10 min depending on sample Farhadian et Effects of Beef (filet) meat, Marinade treatments: BaP BaP Cooking oil as ingredients of al., (2012) marinating commonly used 1) Basic marinade (B): sugar, water, onion, turmeric, 4.78 ± 0.41 B ma rinade treatment for beef for charcoal lemon grass, salt, garlic, coriander and cinnamon. Control: beef 0.11±0.01 satay contributed to a higher grilling of satay, 2) Basic -oil (B-O): (B) plus oil. satay samples 0 hours of concentration of PAH. from local 3) Commercial (Cmr): spices, onion, garlic, salt, sugar, without marinating markets in Seri water and cooking o il. marinating. Preferr ed marinating order to Serdang, Selangor. Modified marinade: (8 hours reduce PAH: Samples were 4) Basic-oil-lemon juice (B-O-L) without chopped into 5) Basic-lemon juice (B-L) marinating) BaP +BbF+F basic- lemon> basic> basic- oil- small pieces 6) Basic-oil-tamarind (B-O-T) 35.8 lemon> basic-o il (about 0.5 cm). 7) commercial -tamarind (Cmr-T) BaP +BbF +F B-L , 4 hours Grilling on a satay -type grill fueled by charcoal for 8 109 of marinating min until the color turned yellowish brown (well B-O, 12 hours done). of marinating 208

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Table 6. (continued). Reference Studied variable Sample Studied grilling conditions Results interval PAH (•g/kg) Conclusion Maximum Minimum Janoszka, Addition of 3 dishes (collars, -Collars: 3-cm diameter.10-cm length. Without Collar meat Onion and garlic as meat (2011) onion and garlic chops and minced Frying for 20 min in a pan preheated to 200 °C. additives: with garlic additives caused a considerable to pork meat chops) made with Addition of 200 mL of water and kept for 1 h at 0.32 ± 0.08 decrease in PAH in the samples and its fried fried pork joint, onion 95 –98°C. Collar meat & grav y of pan fried pork. gravy. (30g/100g meat), -Chops : Onion & garlic mixture spread on the gravy: &onion: The addition of onion led to an garlic (15g/100g meat and kept for 12hours. Frying for 6 min on 1.09 ± 0.06, 0.01 ± 0.002 average decrease of 60% in the meat), without bones each side without fat in a Teflon -coated frying pan 0.08± 0.02 total content of 6 PAH in meat and small fat pieces. preheated to 200 °C. Addition of 100 mL of water Chops & samples and of over 90% in Pan residues obtained and left for 10 min at 95 –98°C. Chops & gravy gravy with gravies. Garlic reduced the from collars and -Minced chops: Minced and mixed meat and 1.61 ± 0.14, onion concentration by 54% in meat chops. vegetables. Chops form: Petri dishes (9 X 1.5 cm) 0.11± 0.01 0.41 ± 0.05 and from 13.5 –79% in gravies. 3 kinds of samples: Grilling in an electric oven at 270 °C for 17 min on 0.01 ± 0.002 No marked effect was found for each side. The grill was 20 cm above the meat. Minced chop: minced chops. a)Without additives -Pan residues obtained from collars and chops: 0.50 ± 0.10 Minced chop b)With onion collected and evaporated nearly to dryness. & onion: c)With garlic 0.38 ± 0.06 Reinik et al., Commercial Estonian home-grilled 2 types of home grilling: 1.8 0.3 (2007) meat products in meat products A) Traditional wood-burning grill: Pork g rilled Chicken Estonia 2001 – Grilled pork, grilled Distance between burning coals and meat: 20 cm over disposable grilled 2005 sausage, grilled Temp. at the surface of the meat: 180°C-240°C. charcoal chicken over chicken. B) Disposable charcoal grill: burning “Less distance than A”. Shorter preparation time. wood 1: !16PAH: naphthalene, acenaphthylene, acenaphthene, fluorene, phenanthrene, anthracene, fluoranthene, pyrene, benz[a]anthracene, chrysene, benzo[b]fluoranthene, benzo[k]fluoranthene, benzo[a]pyrene, indeno[1,2,3- c,d]pyrene, dibenz[a,h]anthracene and benzo[ghi]perylene. n.d: non-detected

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5.3 Operating parameters of smoke generators and liquid smoke.

The main operating parameters affecting PAH formation in friction, steam smoke, touch smoke smoldering and thermostated smoke generators are the temperature of pyrolysis and airflow. Several authors (Hitzel et al., 2013; Pöhlmann et al., 2012, 2013a, 2013b) have conducted a number of detailed studies on the PAH content of Frankfurter-type sausages as a function of smoking conditions. They found that smoking conditions have a significant influence on the PAH content of smoked meat products. PAH content increases with smoke density and ventilator velocity (Pöhlmann et al., 2012). Frankfurter-type sausages smoked by friction smoke generation were found to have the lowest BaP and PAH4 contents (0.03 ± 0.01 •gkg -1 and 0.29 ± 0.11 •gkg -1 , respectively) compared to steam, touch smoke and smoldering methods. Samples produced by smoldering with an intensive smoke density have the highest BaP and PAH4 contents (0.40 •gkg -1 and 2.60 •gkg -1 , respectively) (Pöhlmann et al., 2013a). The most important parameter influencing PAH content is the smoke generation temperature. Temperatures over 600°C must to be avoided to obtain lower PAH contents in smoked meat products (Pöhlmann et al., 2012).

Table 7 shows the PAH content found in meat products with smoke flavorings. In fact, concentrations of up to 2.86 ± 0.39 •g/kg, 3.4 •g/kg and 336.6 •g/kg BaP have been detected in commercial smoke flavorings (Gomaa, Gray, Rabie, Lopez-Bote, & Booren, 1993; Guillén, Sopelana, & Partearroyo, 2000a, 2000b, 2000d; Šimko, Petrík, & Karovi!ová, 1992; Yabiku, Martins, & Takahashi, 1993). The use of different kinds of wood and the material used in the storage container influence the final content of PAH in smoke flavorings. Flavoring obtained from poplar wood presents the highest number and concentrations of both total and carcinogenic PAH compared to that produced with oak, cherry tree, beech, poplar or vine shoot woods. Storing smoke flavorings in polyethylene flasks reduces the concentration of some PAH (Guillén, Sopelana, & Partearroyo, 2000c). The EC has accordingly set 10 •g/kg as the maximum permissible BaP content in smoke flavorings (EC No 2065/2003) and has recently established the EU list of authorized smoke flavoring primary products for use as such in or on foods and/or for the production of derived smoke flavorings (EU, No 1321/2013). Finally, different collaborative projects have been carried out to assess analytical methods for PAH determination in smoke flavorings (Simon, Gómez-Ruiz, & Wenzl, 2010).

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Resultados Table 7. Polycyclic aromatic hydrocarbons (PAH) in meat products with smoke flavorings. Reference Studied variable Sample Studied liquid smoke conditions Results BaP (•g/kg) Conclusion Maximum Minimum Guillén et al., PAH in commercial 5 commercial liquid A: phenol, guaiacol, and syringol derivatives, 2.86± 0.39 n.d BaP was detected in 3 (2000a) liquid smoke flavorings smoke with a small proportion of lignin dimers and samples, but its with different flavorings: A, B, C, D trimers and an insignificant proportion of Commercial Samples D, E concentration did not compositions. and E. carbonyl and carboxyl liquid smoke exceed the 10 •g/kg level derivatives. flavouring fixed by the FAO/WHO . B, C: typical smoke components in similar (sample A) proportions to those found in smoke. D: typical smoke components in similar proportions to those found in smoke (lower than B and C). E: similar to A, but lower concentrations. Guillén et al., PAH in liquid smoke 2 commercial liquid 2 commercial liquid smoke flavorings. 2.86± 0.39 n.d “The flavoring from beech (2000b) flavorings. smoke flavorings. (2 smoke flavorings obtained in the laboratory wood (2 smoke flavorings under controlled conditions by pyrolysis of Commercial Smoke flavoring (hardwood) has higher obtained in the sawdust from beech wood and vine shoots). liquid smoke obtained in concentrations of laboratory under flavoring laboratory carcinogenic PAH than that controlled conditions from vine shoots by pyrolysis of sawdust (softwood)”. from beech wood and vine shoots) Guillen et al., PAH in liquid smoke 5 liquid smoke Round-bottom flask smoke generator made of 0.06± 0.02 n.d in flavorings The flavoring obtained (2000c) flavorings obtained from flavorings obtained in quartz. Poplar stored from oak, cherry from poplar wood presents Different types of wood. the laboratory using dry Pyrolysis was started using a rheostat- controlled in glass flasks tree and vine the highest number and Effect of s torage in sawdust from different heating mantle. shoots stored in concentrations of both polyethylene f lasks on woods: Temperatures reached during the process: glass flasks and in total and carcinogenic their concentrations 530°C (oak), 550°C (cherry tree), 532°C (beech), all flavorings PAH. Storing smoke Oak, cherry tree, beech, 536°C (poplar), and 559°C (vine shoots). stored in flavorings in polyethylene poplar, and Beech, poplar, and vine shoots flavorings were polyethylene flasks reduces the vine sh oots. stored in both glass and polyethylene flasks. flasks except for concentration of some Storage time: 4 years for poplar and vine shoot poplar. PAH. samples, and 2.5 years for beech flavoring.

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Capítulo 4 Table 7. (continued). Reference Studied variable Sample Studied liquid smoke conditions Results BaP (•g/kg) Conclusion Maximum Minimum Guillén et al., PAH in liquid smoke Mixtures either of pure PAH and of Commercial flavorings. 3.1± 0.2 2.6± 0.0 (2000d) flavo rings. smoke flavoring compounds, with the aim of simulating smoke flavorings and, on the other hand, aliquots from two commercial liquid smoke flavorings, A and B. Gomaa et al., PAH in commercial Eighteen commercial liquid smoke Commercial liquid smoke flavorings 3.4 0.1 In liquid smoke flavorings (1993) liquid smoke flavorings flavorings and seasonings and seasonings . and seasonings, total and seasonings PAH concentrations ranged from 6.3 to 43.7 •g/kg, with the carcinogenic PAH ranging from 0.3 to 10.2 •g/kg. Yabiku et al., 11 samples of liquid smoke flavor Liquid smoke flavor. 336.6 0.1 BaP was found in 73%, of (1993) the liquid smoke flavor samples. Three liquid smoke flavor s amples showed levels of BaP above the maximum level recommended by FAO/WHO (10 •g/kg). Šimko et al., Benzo(a)pyrene in liquid Liquid smoke preparations Liquid smoke preparations. 0.8 0.3 (1992) smoke preparations Simon et al., 15 + 1 EU priority PAH Primary Smoke Condensate (PSC). PSC obtained from 6 different 13.4 5 (2010) manufacturers from the smoke flavor industry and mixed in approximately equal amounts. 2 materials evaluated by an inter -laboratory comparison (25 laboratories). n.d: non-detected

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5.4 Position and distance of the product in relation to the heat source.

The distance of the food from the heat source is specified, though not selected as the main variable to study, in several studies on PAH in smoked meat products. The studied distances ranged between 2 and 10 meters (Djinovic et al., 2008; García-Falcón & Simal-Gándara., 2005; Ledesma et al., 2014 ; Škaljac et al. 2014 ).

The study by Pöhlmann et al. (2013b) stands out for researching the influence of the position in the smoking chamber on the final PAH content of meat products, maintaining the remaining variables constant. The positions studied were front, center-front, center-back, and back of the chamber. Results show that meat products placed in the center-back and back have the hig hest and lowest PAH4 (4.57 •g/kg, 3.62 •g/kg) and BaP (1.10 •g/kg, 0.88 •g/kg) contents, respectively.

5.5 Fat content of the product and its evolution during processing.

Similar to the variables of distance and position in the smoking chamber, the fat content is specified in the sample description in several studies on PAH content in smoked meat products (Hitzel et al., 2013; Ledesma et al., 2014, 2015a, 2015b; Pöhlmann et al., 2012, 2013a, 2013b; Škaljac et al., 2014). Several papers may be highlighted for studying this variable: Pöhlmann et al. (2013b), Gomes et al. (2013), García Falcón and Simal Gándara (2005), and Ledesma et al. (2015b). Pöhlmann et al. (2013b) found that BaP content clearly increases with fat content from 0.28 •g/kg in 9.9% fat content to 1.37 •g/kg in 39.1% fat content in smoked Frankfurter-type sausages. Different smoking conditions were adopted in the experiments (different smoke density and ventilator velocity). The combination of intensive (highest) smoke density, the highest ventilator velocity (3000 rpm) and the highest fat content leads to the reported highest BaP contents. In fact, Gomes et al. (2013) concluded that the fat content and smoking regime alone did not influence the total amount of PAH, whereas the casing type has a greater influence on the PAH content of meat products. Studying these two variables, Ledesma et al. (2015b) observed that, during smoking, the fat content flows through natural casings and covers the external surface of the product, making it sticky and moist. Subsequently, when PAH aerosols reach the sticky, wrinkled surface, soot particles are easily captured and adhere to it, damage the casing and start to migrate inside the product. Once captured, the soot particles slowly penetrate inside meat products, accessing the inner part through the lumps of fat. In contrast, the fat

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content of meat products stuffed in synthetic casings remains inside the product. The surface of the casing remains dry, making it non-sticky and smooth, without any affinity for soot particles. Accordingly, a lower amount of soot particles containing PAH (aerosols) was found outside the casing and did not damage its surface. Finally, BaP was found inside meat products stuffed in natural casing (1.71 ± 0.15 mg/kg), while no BaP content was found inside meat products stuffed in synthetic casings (Ledesma et al., 2015b).

5.6 Duration of smoking and direct drying.

Similar to the previously mentioned variables, the duration of smoking is also specified in the sample description in several studies on PAH content in smoked meat products (García- Falcón & Simal-Gándara, 2005; Gomes et al., 2013; Hitzel et al., 2013; Pöhlmann et al., 2012, 2013a, 2013b; Škaljac et al., 2014; Stumpe- V•ksna et al., 2008). Two papers stand out for studying this variable, Djinovic et al. (2008) and Ledesma et al. (2014). In these studies, smoking time ranged from 0 to 18 and 0 to 7 days, respectively, whereas minutes (10-30) are applied in the studies by Pöhlmann et al. (2012, 2013a, 2013b), depending on the type of smoking method employed. Djinovic et al. (2008) and Ledesma et al. (2014) found that the BaP content of meat products clearly increases with smoking time. However, smoking time may not have been the main variable influencing PAH content of smoke meat products. The BaP content of chorizo stuffed in natural casing increases from less than 0.24 •g kg –1 to 0.75 ± 0.05 •g kg –1, finally stabilizing after 5 days of smoking (Ledesma et al., 2014). BaP is mainly deposited in the casing of the product (Andrée, Jira, Schwind, Wagner, & Schwagele, 2010; Djinovic et al., 2008; García- Falcón & Simal-Gándara, 2005; Ledesma et al., 2014; Santos et al., 2011), which works like a barrier to BaP penetration after 5 days of smoking of chorizo (Ledesma et al., 2014). Different BaP contents have been found in similar meat products when applying a similar smoking time: for example, less than 0.48 •g kg –1 after 9 days of smoking of different meat products (Djinovic et al., 2008), and 0.38 •g kg –1 and 0.75 •g kg –1 in chorizo after 3 and 5 days of smoking, respectively (Ledesma et al., 2014).

5.7 Type of casing

It has been found that the greatest amount of BaP is deposited on the meat product casing, only a small amount then migrating into the product (Andrée et al., 2010; Djinovic et al., 2008; García-Falcón & Simal-Gándara, 2005; Ledesma et al., 2014; Santos et al., 2011). In a recent

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study (Pöhlmann et al. (2013b)), lower levels of PAH were found in Frankfurter sausages with peelable cellulose casings compared to natural or collagen casings. CAC recommends cleaning the product by rinsing or immersion in water, as these processes may remove soot and particles containing PAH on the surface of the food (CAC/RCP 68/2009). Some time ago, some authors had already reported that the use of different types of casing (natural and synthetic) has an influence on the PAH content of meat products (Filipovic & Tóth, 1971; Tóth, 1973). A number of studies have recently continued this line of research (Gomes et al., 2013; Pöhlmann et al., 2013b ; Škaljac et al., 2014). Gomes et al. (2013) studied the effect of fat content, casing type and smoking procedures on the PAH contents of Portuguese traditional dried fermented sausages. They found that fermented sausages with collagen casing had the lowest PAH content, showing that the use of synthetic instead of natural casings contributes to reducing PAH levels in smoked meat products. Likewise, Škaljac et al. (2014) studied the influence of smoking under traditional and industrial conditions on the PAH content in dry fermented sausages (“Petrovská klobása”) from Serbia. These authors found that the total levels of the sum of 13 US-EPA PAH were significantly higher in sausages stuffed in natural casings (220 ± 6.30 •g/kg) than in sausages stuffed in collagen casings (31.3 ± 0.55 •g/kg ), even when smoked under the same conditions. Likewise, García-Falcón and Simal-Gándara (2005) found that collagen-based casings act as a better barrier to the penetration of PAH into chorizo meat. Ledesma et al. (2015b) found that this effect is caused by differences in the physical properties of the two types of casing. To sum up, the high porosity (66.8%) of natural casings allows the fat content to flow through the casing and cover its external surface, making it sticky and moist. This effect, added to the product’s wrinkled surface, allows soot particles to be easily captured and adhere to it. These particles subsequently damage the casing and start to migrate inside the product. In contrast, the low porosity (16.6%) of synthetic casings makes the fat content remain inside the product. The surface of synthetic casing is dry, non-sticky and smooth, showing no affinity for soot particles. Moreover, the smaller size of the pores prevents any small amount of coal tars from penetrating the product. More differences were also found between casings. Besides pore characteristics, the diffusivity of the compounds in the continuous phase is also an important factor (Díaz, Vega, & Coca, 1987). The average pore diameter (599.8 nm) and the interstitial porosity (47.6%) of natural casing are considerably higher than those of collagen casing (48.2 nm, 25.9%). However, the total pore area of both types of casing is similar (Ledesma et al., 2015b). The typical size of smoke particles (containing PAH) ranges between 200nm and 400 nm, while the total values range between 50 nm and 1000nm (CAC, 2009). Thus, smoke particles are larger than collagen casing pores and

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smaller than those of natural casing. This fact explains the penetration of smoke particles in natural casings, as opposed to collagen casings. Casing type is a variable that has an important effect on the PAH content of smoked meat products and should be considered in meat product manufacturing in order to obtain healthier products.

5.8 Evaluation of variables

As shown, a great number of variables alter the PAH content of smoked meat products. It may thus be very difficult to control all these variables, although important conclusions can be drawn from the research studies reported here. In order to prevent PAH contamination of smoked meat products, three variables seem to have a greater influence than others. These variables are: a) the type of casing, b) the smoking method, which may be direct (traditional smoking) or indirect (friction, smoldering, thermostated plates, liquid smoke), and c) the temperature of smoke generation. A smoke generation temperature below 600°C should be applied to prevent the formation of PAH. The use of synthetic instead of natural casings prevents the penetration of PAH inside products smoked by any method. Casings should be removed by consumers before consuming meat products; peelable cellulose casings, which have already been implemented by some producers, can contribute in this respect. Indirect smoking systems (such as friction smoke generation) can highly reduce the PAH content of meat products, while the use of liquid smoke could be a good alternative if the fractionation and purification processes are really improved and optimized to guarantee the absence of toxic and carcinogenic impurities and the most appropriate wood is used. Moreover, a final step involving the storing of smoke flavorings in polyethylene flasks helps to reduce PAH content. Other variables such as the use of different kinds of wood (never using gasoline, treated woods or wasted oil) and controlling the smoking time and fat content can help reduce the final PAH content of the product. Research focused on methods for reducing the tar content could be applied to improve the design of the meat product smoking process and smoking chambers to prevent PAH contamination of meat products. Moreover, microwave preheating, wrapping in aluminum, acidic marinades and the addition of onion and garlic as meat additives can help to reduce the PAH content of grilled meat products considerably. The PAH content of smoked food could be minimized taking into account the variables recommended by CAC/RCP 68/2009 and studied by several researchers. This is especially important in those developing countries were the cold chain has not been established yet (Ogbadu, 2014; FAO-Thiaroye, 2015). Considering this, The National Training Centre for

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Fisheries and Aquaculture Technicians (CNFTPA) of Senegal in collaboration with FAO have designed and developed the FTT-Thiaroye technique. This technique and its diffusion help to minimize the PAH content of the smoked food in the developing countries (FAO-Thiaroye, 2015). More research and application of the CAC/RCP 68/2009 code will improve the safety of smoked food in the world.

6. Conclusions

Meat and meat products are an important component of human diet, especially due to their high protein content. Smoking has traditionally been used to prolong the shelf life of meat products, and is it still applied as preserving method in developing countries. Smoking is still used in developed countries to improve food flavor, color and smell. However, undesirable carcinogenic substances are produced in this process such as polycyclic aromatic hydrocarbons (PAH). The formation of PAH during smoking can be explained by means of biomass conversion. PAH are tertiary tar products formed via biomass gasification and pyrolysis, starting above 750°C. Tar aerosols are produced, pass through the smoking chamber and are deposited on meat products. Meat products can be contaminated by PAH in different ways, as a result of pre- contamination of ingredients, technological processes different to smoking, and fat falling from other products in the smoking chamber. The current data on BaP and PAH4 contents in meat products around the world show that, while the new legal limits are respected in the majority of cases, they are still greatly exceeded in others. According to the literature reviewed in this paper, 3 out of the 10 variables recommended by CAC/RCP 68/2009 seem to have a greater influence on preventing PAH contamination: the temperature of smoke generation, the type of casing and the smoking method (direct or indirect). A smoke generation temperature below 600°C should be applied to prevent the formation of PAH. The use of synthetic instead of natural casings prevents PAH from penetrating inside products. Indirect smoking systems (e.g., friction or liquid smokes) can highly reduce the PAH content of meat products. On the other hand, the control of other variables such as wood (never gasoline, treated woods or wasted oil), marinating, smoking time and fat content can help to reduce the final PAH content of smoked meat products. Finally, meat product smoking can be adapted to obtain PAH contamination levels below the new European limits.

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Acknowledgement

The authors would like to acknowledge the staff at the El Hórreo Healthy Food S.L. company, who contributed their knowledge and expertise in meat product smoking.

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Discusión•general• • • • • • • • • Discusión general

5. DISCUSIÓN GENERAL

5 .1 DISCUSIÓN DE RESULTADOS

La presente tesis doctoral ofrece resultados científicos relevantes que permiten controlar y reducir la contaminación de productos cárnicos ahumados a modo directo por las sustancias cancerígenas hidrocarburos aromáticos policíclicos (HAP). La aplicación de estos resultados es especialmente importante para la creciente industria cárnica de los países desarrollados (FAO, 2014a), y las comunidades de los países en desarrollo (FAO-Thiaroye, 2015; Ogbadu, 2014; Vaz- Velho, 2003) que continúan utilizando el ahumado directo como método productivo y de conservación de los alimentos.

Haciendo una revisión ( Capítulo 4.5 ) se encontró que algunas investigaciones recientes reportan valores de BaP por debajo del nuevo límite (2 •g/kg) establecido por el Reglamento (UE) No 835/2011, e introducido el 1/09/2014. Por ejemplo no superan este valor algunos productos cárnicos ahumados de la República de Korea (Chung et al., 2011), España (Martorell et al., 2010), Italia (Purcaro et al., 2009) o Portugal (Santos, Gomes, & Roseiro, 2011). Sin embargo, todavía se reportan también valores muy elevados, como 36,9 •g/kg en jamón ahumado de Suecia (Wretling, Eriksson, Eskhult, & Larsson, 2010), 10,02 •g/kg en productos de cerdo ahumado de Sudáfrica (Olatunji et al., 2014), 6,98 •g/kg en productos cárnicos bovinos en Malasia (Jahurul et al., 2013), 31,20 •g/kg en carne ahumada de Estonia (Reinik et al., 2007) y 17,63 •g/kg en productos de cerdo ahumado de Alemania (Jira, Ziegenhals, & Speer, 2008).

Estos datos indican que todavía es necesario establecer medidas para prevenir la contaminación por HAP de productos cárnicos ahumados. Para ello, la presente tesis doctoral se ha basado en las normativas y recomendaciones oficiales, como el “Código de prácticas para reducir la contaminación por hidrocarburos aromáticos policíclicos (HAP) en los alimentos producidos por procedimientos de ahumado y secado directo (CAC/RCP 68-2009), de la Comisión del Codex Alimentarius (CCA), las regulaciones europeas N o 1881/2006 y N o 835/2011, así como en el estudio del estado del arte a nivel científico.

Las normas y la comunidad científica indican que la contaminación por HAP de productos cárnicos ahumados está afectada por un elevado número de factores, englobados por el Codex

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Alimentarius en 10 variables del proceso. Estas variables han sido estudiadas por varios investigadores actuales. Haciendo una revisión de todas ellas ( Capítulo 4.5 ), se pudo concluir que 3 son las más importantes para controlar la presencia de HAP en los productos: la temperatura de generación de humo, el tipo de tripa, y el método de ahumado (directo (ahumado tradicional) o indirecto (ahumado por fricción, fuego sin llama, autocombustión o uso de humo líquido)). Se deberían aplicar temperaturas por debajo de los 600ºC para prevenir la formación de HAP (Pöhlmann et al., 2012). El uso de tripas sintéticas previene la penetración de los HAP en los productos cárnicos ahumados por cualquier método, hecho confirmado en esta tesis en ahumado directo de chorizo, por García Falcón y Simal Gándara (2005), Gomes, Santos, Almeida, Elias, y Roseiro (2013), Pöhlmann et al. (2013a) y Škaljac et al. (2014). Cualquier tipo de tripa debería ser retirada antes del consumo de un producto cárnico ahumado, por lo que el uso de tripas pelables de celulosa es muy aconsejado (Pöhlmann et al., 2013a). Los sistemas indirectos de ahumado (como el ahumado por fricción) pueden reducir en gran medida la cantidad de HAP de los productos (Pöhlmann et al., 2013b), mientras que el humo líquido es una buena alternativa si los procesos de fabricación y purificación han sido realmente optimizados para garantizar la ausencia de impurezas cancerígenas y tóxicas, y la madera utilizada ha sido correctamente seleccionada. Además la conservación de humos líquidos en contenedores de polietileno ayuda a reducir el contenido de HAP (Guillén, Sopelana, & Partearroyo, 2000). Otras variables como el uso de distintos tipos de madera (nunca utilizar gasolina, maderas pretratadas o aceite usado) (Hitzel, Pöhlmann, Schwägele, Speer, & Jira, 2013; Stumpe-V!ksna et al., 2008) o el control del tiempo de ahumado, tal y como se ha estudiado en esta tesis, y por Djinovic, Popovic, y Jira (2008), la posición del alimento respecto a la fuente de calor (Pöhlmann et al., 2013a), y la grasa del producto y su evolución, tal y como se ha estudiado en esta tesis, y por García Falcón y Simal Gándara (2005), Gomes et al. (2013) y Pöhlmann et al. (2013a), puede ayudar a reducir la cantidad de HAP del alimento final. Además el precaliento con microondas, envoltura en aluminio (Farhadian, Jinap, Hanifah, & Zaidul, 2011), marinados ácidos (Farhadian, Jinap, Faridah, & Zaidul, 2012) y la adición de cebolla y ajo como aditivos (Janoszka, 2011) podría ayudar a reducir el contenido de HAP en el caso de productos cárnicos ahumados y hechos a la parrilla.

Considerando todos estos aspectos, la presente tesis doctoral se ha enfocado en el estudio de la contaminación por HAP durante el ahumado a modo directo de chorizo en el Principado de Asturias. Para ello, en la presente tesis doctoral, además de estudiar otras, se ha seleccionado una de las 3 variables más importantes, y la única que se puede controlar utilizando

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el ahumado directo tradicional del chorizo asturiano: el tipo de tripa. Así mismo, se ha centrado en el hallazgo de los motivos, que expliquen científicamente, el proceso de transporte y penetración de HAP en los productos, para proponer métodos de prevención de la contaminación de estos alimentos por los cancerígenos HAP.

En particular, mediante el primer estudio ( Capítulo 4.1 ), se puso a punto un método para determinar BaP en chorizo ahumado. Este método está basado en la combinación de las técnicas de pretratamiento de muestra modernas, sonicación y SPE, que garantizan la reducción de consumo de disolventes y tiempo de análisis respecto a las técnicas clásicas, como la combinación de Soxhlet y GPC. La aplicación de este método en 16 chorizos ahumados fabricados por 16 empresas diferentes del Principado de Asturias, obtenidos en establecimientos comerciales, confirmó que 5 de los 16 productores sobrepasaron el nuevo límite máximo de BaP (2 •g/kg) en productos cárnicos ahumados establecido por la regulación europea Nº 835/2011, e introducido el 1/09/2014. El rango de BaP encontrado fue 0,38 –3,21 g/kg. Se encontró que el contenido de humedad y BaP de las muestras no estaba correlacionado. En definitiva, algunos productos contenían un elevado contenido tanto de BaP como de humedad, relevando la baja calidad del ahumado como sistema de secado. Este hecho enfatiza la necesidad de facilitar recomendaciones a los productores.

En el segundo estudio ( Capítulo 4.2 ), se confirmó que utilizando un sistema de ahumado controlado, bajo las variables “tipo de combustible, ingredientes del producto, distancia entre el alimento y fuente de calor” constantes , y estudiando la variable “tiempo de ahumado”, la cantidad de BaP del chorizo (sin tripa) aumenta desde menos de 0, 24 •g/kg, antes de ahumar, hasta 0, 75 •g/kg, estabilizándose entre los 5 y 7 días de ahumado. El contenido de humedad disminuye desde el 49,9% al 31,3% en este periodo. Otros investigadores (Djinovic et al., 2008) han encontrado recientemente una tendencia similar de aumento del contenido de BaP con el tiempo de ahumado, si bien con valores menores a los hallados en esta tesis doctoral, incluso con mayor tiempo de ahumado. Además, en nuestro trabajo, se realizó un estudio del contenido de BaP en distintas profundidades de chorizo, ya que el Codex Alimentarius proponía, como tratamiento posterior al ahumado, el rasurado de la superficie del alimento para eliminar el hollín y las partículas que contienen HAP (CCA, 2009). En efecto, varias referencias científicas actuales estaban confirmando este hecho en productos de todo el mundo (Andrée, Jira, Schwind, Wagner, & Schwagele, 2010; Djinovic et al., 2008; García-Falcón & Simal-Gándara, 2005; Santos

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et al., 2011). En este trabajo se realizó un estudio completo y novedoso sobre la penetración de BaP en diferentes profundidades en el chorizo asturiano ahumado. Se encontró que la cantidad de BaP disminuye y el contenido de humedad aumenta progresivamente desde la superficie (profundidad D1: 20, 0 ± 1.1 •g/ kg, 12,4% ± 1,4%), hasta el interior del producto (profundidades D2: 1, 63 ± 0.11 •g/kg, 29.2% ± 1,5%; D3: 1,12 ± 0, 10 •g/kg , 35,5% ± 0,3%; y D4: 0,88 ± 0,10 •g/kg , 33,8% ± 1,3%). Estos datos permitieron proponer un mecanismo esquemático que explica la penetración de BaP y el secado del chorizo durante el proceso de ahumado. Resultó de especial relevancia encontr ar un contenido de BaP de 20 •g/kg en la superficie del producto. Por ello, los productores deben supervisar el estado de las tripas durante el procesado. Si la tripa se rompe los HAP pueden entrar más fácilmente en el interior del producto. Por otro lado, se aconseja a los consumidores que eliminen la tripa natural ( también llamada “piel”) del cho rizo ahumado, antes de su consumo.

Dada la relevancia de los resultados obtenidos en el segundo estudio, relacionados con el elevado contenido de BaP en la superficie del producto, el tercer estudio ( Capítulo 4.3 ) se enfocó en evaluar el flujo de este compuesto a través de la tripa, que funciona como una membrana. Varias referencias científicas habían dilucidado en los años 70 que el uso de distintos tipos de tripa tendría influencia en el contenido de HAP de los productos cárnicos (Filipovic & Toth, 1971; Toth, 1973), siendo por lo tanto el punto de mira de las investigaciones actuales (García-Falcón & Simal-Gándara, 2005; Gomes et al., 2013; Pöhlmann et al., 2013a; Škaljac et al., 2014), las cuales coincidieron en el tiempo con las nuestras. Por tanto, en esta tesis se estudió el contenido de BaP durante el ahumado de chorizo asturiano embutido en tripa natural (ChN) y sintética, de colágeno (ChS). Teniendo en cuenta los resultados obtenidos en el segundo estudio, se diseñó paralelamente un sistema novedoso de tripa llenada con aceite, que permitiera eliminar las barreras causadas por las distintas profundidades encontradas en el producto cárnico, siendo las grasas un medio idóneo para la dilución y concentración de los compuestos de interés, ya que los HAP son compuestos lipofílicos (Vázquez Troche et al., 2000), que se disuelven en las grasas en estado líquido de manera uniforme. En ambos sistemas (chorizo y aceite), se encontró que el BaP no atravesaba la tripa sintética. Al contrario, se encontró un contenido de 2,16 ± 0, 12 •g/kg y 1,71 ± 0,15 •g/kg en el interior de los sistemas de aceite y ChN, respectivamente. Por otro lado, confirmando los resultados del estudio previo, se encontró un contenido de 19,9 ± 2,9 • g/kg en el exterior de la tripa natural. Nuestros resultados fueron corroborados por otros autores que realizaron estudios simúlatenos en el tiempo, encontrando que las tripas sintéticas previenen la

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penetración de HAP en otros productos cárnicos ahumados (Gomes et al., 2013; Pöhlmann et al., 2013a; Škaljac et al. , 2014). Dado que el hecho estaba confirmado, se planteó como estudio en la frontera del conocimiento encontrar una explicación científica que justificara estos hechos. Para ello se caracterizaron las diferencias físicas entre los 2 tipos de membrana (tripa natura y de colágeno), y la evolución de los productos cárnicos durante el tiempo de ahumado. Los estudios planteados fueron la determinación de la porosidad de las tripas, y caracterización morfológica de ambos tipos de tripas sin ahumar y ahumadas, y 48 productos embutidos en ellas durante el proceso de ahumado, de 0 a 10 días. Para la caracterización morfológica se utilizaron las técnicas microscopía electrónica de barrido (SEM) y microscopía de fluorescencia óptica. La mayor dificultad fue la imposibilidad de determinar la porosidad de la tripa natural en estado húmedo, pues el dispositivo experimental (porosímetro) no permite la medida de muestras húmedas. Para resolver este problema se creó un sistema novedoso de secado lento de ambos tipos de tripa, que no dañase su estructura y sometiera a ambas a las mismas condiciones. Los resultados obtenidos fueron los más novedosos a nivel científico de la presente tesis doctoral y permitieron proponer un mecanismo que explica porqué el BaP atraviesa la membrana natural y no la sintética, basado a demás en una de las variables fijadas por el Codex Alimentarius: lo que sucede al contenido graso del producto durante el ahumado. Además, el mecanismo que lo explica fue completado mediante el estudio bibliográfico del proceso tecnológico de ahumado y el estado del arte sobre contaminación de HAP en productos cárnicos, lo cual dio lugar a la redacción de un review ( Capítulo 4.5 ). A modo de resumen, del estudio bibliográfico se concluye que los productos cárnicos pueden ser “precontaminados” por HAP debido a la contaminación ambiental de las materias primas (CCA, 2004, 2009), en segundo lugar, los procesos tecnológicos diferentes al ahumado pueden contribuir al contenido total de HAP de los alimentos (CCA, 2009; Chung et al., 2011; Kazerouni, Sinha, Hsu, Greenberg, & Rothman, 2001). Finalmente, durante el proceso tecnológico de ahumado, la combustión incompleta y pirólisis de biomasa a temperaturas superiores a 750 ºC da lugar a la formación de compuestos de tar terciarios, los cuales contiendo los HAP (Basu, 2010, chap.4), son transportados en forma de aerosol hasta los productos cárnicos dentro de la cámara de ahumado. Por otro lado, en los experimentos realizados en esta tesis doctoral, se encontró que las tripas naturales se caracterizan por su rugosidad y capacidad para atrapar cuerpos pequeños, mientras que las tripas de colágeno se caracterizan por formar una red-barrera de fibras sin capacidad para capturar cuerpos pequeños. Una vez dentro de la cámara de ahumado, los chorizos embutidos en distintos tipos de tripa, natural (ChN) y sintética (ChS), se calientan y comienzan a secarse. La elevada porosidad de las tripas naturales (66,8%)

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hace que incluso las materias primas crucen sus poros. Durante el ahumado, estos chorizos (ChN) se calientan, se hinchan y la grasa fluye a través de la tripa natural, convirtiendo la superficie de los chorizos en un medio pegajoso donde las partículas de humo son fácilmente capturadas y adheridas, y finalmente migran a través de los poros de gran diámetro (599,8 nm) de la tripa natural hacia el interior del producto. Al contrario, la morfología lisa y la baja porosidad (16,6%) de las tripas sintéticas hace que éstas funcionen como una barrera. Los chorizos embutidos en esta tripa (ChS) no se hinchan, y la grasa permanece en el interior del producto, mostrando una superficie seca, no pegajosa y lisa, con menos afinidad a capturar las partículas de humo que contienen HAP, cuya dificultad de penetrar en el producto se incrementa debido al pequeño diámetro de los poros (48,2 nm). Estos hechos explican los resultados sobre penetración de BaP a través de la tripa natural encontrados en nuestros estudios previos, así como por García-Falcón y Simal-Gándara (2005), Gomes et al. (2013) y Škaljac et al. (2014). El código de prácticas para reducir la contaminación por HAP en los alimentos producidos por procedimientos de ahumado y secado directo, (CCA, 2009), indica que el humo consta de particulados líquidos y sólidos suspendidos en una fase gaseosa, formados por numerosos componentes, entre los que se encuentran los HAP. Según el código, se estima que el tamaño de las partículas del humo oscila entre 0 ,2 y 0,4 •m (o un tamaño tan pequeño como 0,05 a 1 •m ), las cuales constituyen el 90% del peso total del humo. Estos datos justifican la teoría expuesta en esta tesis. Así, el BaP es un HAP transportado por las partículas del humo hasta los productos cárnicos. Estas partículas no penetran a través la tripa sintética, dado que el tamaño de las partículas más pequeñas (50 nm), es mayor que el diámetro medio de los poros de la tripa sintética (48,2 nm); sin embargo si penetran a través de la tripa natural, ya que el tamaño medio de las partículas (entre 200 y 400 nm) es menor que el tamaño medio del diámetro de los poros de la tripa natural (599,8nm).

Por último, se planteó determinar si las propiedades físicas y organolépticas de los chorizos son adecuadas en ambos casos, embutidos en tripa natural (ChN) y en sintética (ChS). Por tanto, en el último estudio ( Capítulo 4.4 ), se determinaron las características físicas (textura, color y humedad) de 48 chorizos asturianos, embutidos en distintos tipos de tripa, durante el tiempo de ahumado (0 a 11 días). Además también se evaluó el proceso de adherencia y penetración de las partículas en distintas profundidades del producto. A modo de resumen, la tendencia general encontrada es que las propiedades son adecuadas en los dos casos, sin embargo los productos se oscurecen y endurecen significativamente antes (p<0.05) si son embutidos en la tripa sintética. Además, aunque las diferencias no fueron significativas (p>0.05),

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la chorizos embutidos en esta tripa podrían secarse antes, pues mostraron siempre menor contenido de humedad. En este estudio se demostró que una vez que las partículas de humo llegan y penetran a través de la tripa natural, se colocan en las cavidades de la masa del producto, las cuales se cierran durante el secado, trasladándolas hasta el interior del producto (hecho que explica encontrar BaP en el interior del producto en el capítulo 4.2 ). Además, los chorizos embutidos en tripa sintética muestran una morfología homogénea que facilita la estandarización del producto, característica demanda por los distribuidores y consumidores. Estos resultados demuestran que las tripas sintéticas son capaces de reducir el tiempo de procesado, y garantizar la estandarización del producto, validando estos factores sugeridos por los fabricantes de tripas sintéticas (CCTA, 2015; DEVRO, 2015; FIBRAN, 2015; VISCOFAN, 2015), y además protegen el producto de la contaminación por HAP, con la ventaja de seguridad alimentaria que esto implica. Es importante señalar que el uso de tripas naturales es correcto y ofrece buenas propiedades físicas y tecnológicas al producto, tal y como sugieren sus fabricantes (ENSCA, 2015; FEDECARNE, 2013; INSCA, 2015), siempre y cuando no sea sometido a un proceso tecnológico de ahumado directo intenso, prolongado e incontrolado, que los contamine con HAP. El control de las variables del proceso de ahumado, de acuerdo a las recomendaciones del CCA y los resultados obtenidos por muchos investigadores, permite el uso de las tripas naturales para la producción de cárnicos ahumados.

Finalmente, los datos actuales de contenido de BaP y HAP4 en productos cárnicos y alimentos ahumados de todo el mundo son todavía muy elevados en algunos casos, especialmente donde se aplica el ahumado directo, en las pequeñas empresas cárnicas de los países desarrollados y más extensamente en los países en desarrollo. Las técnicas de ahumado pueden controlarse para alcanzar valores por debajo de la normativa europea, que garanticen la seguridad alimentaria de estos productos de importante valor económico y nutricional para la dieta humana.

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6.•CONCLUSIONES•

El trabajo expuesto en la presente tesis doctoral permite obtener las siguientes conclusiones:

El ahumado directo es una de las técnicas de conservación de alimentos más antiguas, y ha tenido un rol importante en la evolución y la dieta humana. Los principales tipos de alimentos ahumados son las carnes, los pescados, los quesos, algunas especies y bebidas. Esta técnica ha sido remplazada en algunos países desarrollados por modernas técnicas de preservación de alimentos frescos y tecnologías optimizadas de ahumado. Sin embargo sigue siendo utilizado por algunas pequeñas empresas de los países desarrollados, y más extensamente en los países en desarrollo.

Durante el ahumado directo se forman numerosos compuestos, que ejercen efectos deseados y no deseados sobre los alimentos. Entre los efectos deseados destacan la prolongación de la vida útil y la mejora de las propiedades organolépticas, color, textura y flavor, de los alimentos. Entre los efectos no deseados destaca la contaminación por sustancias tóxicas y cancerígenas, como los hidrocarburos aromáticos policíclicos (HAP), las n-nitrosaminas, las aminas aromáticas heterocíclicas y los •-carbonilos. Las investigaciones se han focalizado en los HAP debido a su elevada actividad cancerígena, y especialmente en productos cárnicos ahumados, ya que se ha encontrado que son los principales contribuyentes de HAP en la dieta, y que contienen los niveles más elevados de HAP.

El control de las variables del proceso tecnológico de ahumado, recomendadas por el Codex Alimentarius CAC/RCP68/2009, permite minimizar la contaminación de los productos cárnicos por HAP durante el proceso, obteniendo valores de benzo(a)pireno (BaP) (un marcador de la presencia y efecto de los HAP en alimentos) por debajo del contenido máximo permitido, reducido a 2 !g/kg por la Regulación Europea No. 835/2011, e introducido el 1 de septiembre de 2014.

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La combinación de las técnicas de pretratamiento de muestras, sonicación y extracción en fase sólida (SPE), y la determinación analítica mediante cromatografía de gases/espectrometría de masas (GC/MS), es un método de análisis óptimo para la determinación del contenido de BaP en chorizo ahumado a modo directo, implicando además una reducción del consumo de disolventes, generación de residuos, tiempo de operación y complejidad del montaje, respecto a técnicas clásicas, como la combinación de soxhlet y cromatografía de permeación de gel (GPC).

El rango de contenido de BaP encontrado en muestras de chorizo ahumado (medido sin tripa) elaborado por empresas del Principado de Asturias fue de 0,38 –3,21 •g/kg. Cinco de las 16 muestras superan el contenido máximo legal de BaP. No se encontró correlación entre el contenido de BaP y la humedad, relevando la baja calidad del ahumado directo, utilizado como método de secado. Por tanto, las empresas del Principado de Asturias deben optimizar su método de ahumado directo, para obtener chorizos de elevada calidad y seguridad alimentaria.

El incremento del tiempo de ahumado, desde 0 a 7 días, produce efectos contrarios entre el contenido de BaP y la humedad del chorizo, medido sin tripa. Mientras que el contenido de humedad decrece (desde 49,9% ± 3,2% hasta 31,3% ± 1,2%), el contenido de BaP aumenta con el tiempo de ahumado (desde menos de 0,24 • g/kg hasta 0,75 ± 0, 05 • g/kg), y finalmente se estabiliza desde los 5 hasta los 7 días de ahumado, con un valor por debajo del límite legal europeo.

La selección y descripción de la profundidad de la muestra tomada es un parámetro fundamental al definir el contenido de BaP del chorizo ahumado a modo directo, embutido en tripa natural. El contenido de BaP decrece, y la humedad aumenta, progresivamente desde la tripa (20,0 ± 1, 1 • g/kg, 12,4% ± 1,4%) hasta el interior del producto (hasta el núcleo: 0,88 ± 0, 10 • g/kg, 33,8% ± 1,3%). El contenido de BaP en la tripa del chorizo es 10 veces superior al límite legal y casi 20 veces mayor que en el núcleo. Por tanto, se recomienda retirar la tripa del chorizo ahumado, antes del consumo. Con el aumento del tiempo de ahumado los componentes del humo podrían obstruir los poros de la tripa, generando una barrera para la penetración de mayor

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contenido de BaP. Sin embargo, la degradación de las tripas podría producir el efecto contrario.

Durante el ahumado directo, tienen lugar la pirolisis y la combustión incompleta de la biomasa, dando lugar a la formación de tar. Cuando la temperatura alcanza los 750ºC se comienzan a formar los productos de tar terciaros, entre los cuales se encuentran los HAP. En la cámara de ahumado, los HAP son transportados hacia los productos cárnicos a través de aerosoles de tar, que forman parte del humo. Al mismo tiempo, los chorizos se calientan y comienzan a secarse. Las tripas natural (de cerdo) y sintética (de colágeno) presentan diferencias físicas que producen un comportamiento diferente de los chorizos embutidos en ellas.

La morfología irregular, la elevada porosidad (66,84%), y el gran diámetro de los poros (599,8 nm diámetro medio) de la tripa natural, hace que la grasa del chorizo fluya a través de ella, el chorizo se hinche, y su superficie se convierta en un material pegajoso donde se adhieren fácilmente las partículas del humo, cuyo tamaño, de acuerdo al Codex Alimentarius, oscila entre los 200 y 400 nm. Por ello, las partículas penetran a través de los poros de la tripa, se colocan en las grandes cavidades de la masa cruda, y comienzan a migrar hacia el interior del producto a través de la grasa, y la compactación de la masa durante el secado, contaminado el interior del chorizo con BaP.

Por el contrario, la tripa de colágeno tiene una morfología lisa y baja porosidad (16,6%). La grasa permanece en el interior del chorizo embutido en ella, mostrando una superficie seca y dura, con menor afinidad a capturar las partículas de humo, cuya posibilidad de penetrar en el producto está limitada por el pequeño diámetro de sus poros (48,2 nm), impidiendo la penetración y contaminación del producto con BaP.

Durante el ahumado directo, los chorizos embutidos en tripa de colágeno se oscurecen y endurecen significativamente (p<0,05) antes, y se secan más rápidamente (aunque las diferencias no fueron significativas, p>0,05), que los embutidos en tripa natural. Los chorizos embutidos en tripa de colágeno muestran una morfología homogénea, que facilita la estandarización del producto, característica demanda por las grandes empresas distribuidores y de platos precocinados. Los chorizos embutidos en tripa natural presentan una morfología irregular, característica demandada por el consumidor de

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producto tradicional. Las tripas natural y de colágeno confieren al chorizo buenas propiedades de textura, color y humedad, pero la tripa de colágeno permite reducir el tiempo de procesado y prevenir la contaminación por BaP en el interior del producto, si es ahumado a modo directo.

El contenido de BaP en productos cárnicos ahumados mediante diferentes técnicas, reportado por la comunidad científica, todavía sobrepasa en algunos casos el límite reglamentario. De la revisión de las referencias científicas, se puede concluir que 3 de las variables recomendadas por el Codex Alimentarius tienen mayor influencia para prevenir la contaminación por HAP de productos cárnicos ahumados: la temperatura de generación del humo, el tipo de tripa y el método de ahumado (directo o indirecto). Se deben aplicar temperaturas por debajo de 600ºC para evitar la formación de HAP, el uso de sistemas de ahumado indirecto, como el ahumado por fricción o el humo líquido, minimiza el contenido de BaP generado, y el uso de tripas sintéticas previene la penetración de los HAP en los productos cárnicos ahumados por cualquier método. El control de otras variables, como la madera, el tiempo de ahumado, la posición del alimento respecto a la fuente de calor, y la grasa del producto y su evolución, puede ayudar a reducir la cantidad de HAP del producto final.

El proceso de ahumado directo de chorizo debe, y puede, ser optimizado mediante el control de variables recomendadas por el Codex Alimentatius, como el tiempo de ahumado, la selección del tipo de tripa y su eliminación antes del consumo, para minimizar la contaminación por BaP, y respetar los nuevos límites establecidos por la regulación europea.

La aplicación de tecnologías modernas de ahumado y ahumado directo de un modo controlado, permite mantener los efectos deseados y prevenir los no deseados del ahumado directo. Esto es importante en algunas pequeñas empresas alimentarias de los países desarrollados, y especialmente en los países en desarrollo, donde el ahumado directo es todavía el principal método de conservación de alimentos.

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•••••••••••••••••••••••••••••••••••••••••••• Bibliografía• • • • • • • Bibliografía

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Reglamento (CE) Nº 1881/2006 de la Comisión de 19 de diciembre de 2006 por el que se fija el contenido máximo de determinados contaminantes en los productos alimenticios. Obtenido el 25 de enero de 2015, de: http://www.boe.es/doue/2006/364/L00005-00024.pdf

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8.•ANEXOS•

8.18 .1 DIFUSIÓN DE LA TESIS DOCTORAL

8.1.1 ARTÍCULOS CIENTÍFICOS

Ledesma, E., Rendueles, M., & Díaz, M. (2015). Spanish smoked meat products: Benzo(a)pyrene (BaP) contamination and moisture. Journal of Food Composition and Analysis, 37, 87 –94.

Ledesma, E., Rendueles, M., & Díaz, M. (2014). Benzo(a)pyrene penetration on a smoked meat product during smoking time. Food Additives and Contaminants, 31, 1688-1698.

Ledesma, E., Rendueles, M., & Díaz, M. (2015). Characterization of natural and synthetic casings and mechanism of BaP penetration in smoked meat products. Food Control, 51, 195-205.

Ledesma, E., Laca, A., Rendueles, M., & Díaz, M. Texture, colour and optical characteristics of a meat product depending on smoking time and casing type. Enviada a la revista Food Chemisty (Elsevier) para su evaluación, bajo revisión.

Ledesma, E., Rendueles, M., & Díaz, M. Contamination by polycyclic aromatic hydrocarbons in the meat products smoking: Processes and Prevention. Enviado a la revista Food Control (Elsevier) para su evaluación, bajo revisión.

Ledesma, E., Rendueles, M., & Díaz, M. Smoked Food. Capítulo de libro de Enciclopedia “Biotechnology” de Elsevier. Pendiente de envío.

Figura 8.1. Revistas científicas de difusión de la presente tesis doctoral.

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8.1.2 COMUNICACIONES A CONGRESOS

E. Ledesma, M. Rendueles, M. Díaz. Physical characterization of natural and synthetic casings for polycyclic aromatic hydrocarbons (PAH) prevention in smoked meat products (keynote). II International Congress of Chemical Engineering (ICCE) of the Spanish's leading organization for Chemistry and Chemical Engineering professionals (ANQUE). Madrid Figura 8.2. ICCE. (Spain). July 1-4, 2014.

E. Ledesma, J.S.Monte, J.Monte, M.Rendueles, M.Díaz. Optimisation of analytical methods for BaP determination in smoked meat products (póster). 11th International Nutrition & Diagnostics Conference (INDC). Brno (Czech Republic). August 28-31, 2011. Figura 8.3. INDC.

Figura 8.4. Presentación de poster INDC. Brno, República Checa, 28-31 de agosto, 2011.

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8.2 INFORME DEL FACTOR DE IMPACTO DE LAS PUBLICACIONES PRESENTADAS

Los artículos que conforman la presente memoria de tesis doctoral, han sido publicados en revistas científicas incluidas en el Science Citation Index (Thomson Reuters), cuyos factores de impacto, y su posición en el área, en función del mismo, se presentan a continuación:

Food Additives and Contaminants, part A :

-Factor de impacto (2013): 2,341.

-Factor de impacto de los últimos 5 años (2013): 2,621.

Food Control :

-Factor de impacto (2013): 2,819.

-Factor de impacto de los últimos 5 años (2013): 3,038.

Journal of Food Composition and Analysis :

-Factor de impacto (2013): 2,259.

-Factor de impacto de los últimos 5 años, (2013): 2,799.

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8.3 NOMENCLATURA

8.3.1 ACRÓNIMOS

AETRIN Asociación Española de Tripa Natural .

AICR American Institute for Cancer Research.

ANICE Asoci ación Nacional de Industrias de la Carne de España.

ASINCAR Centro Tecnológico Agroalimentario “Asociación de Industrias Cárnicas del Principado de Asturias” .

BOE Boletín Oficial del Estado .

CCA Comisión del Codex Alimentarius .

CCTA Col lagen Casings Trade Association.

CE Comisión Europea.

EEUU Estados Unidos de América.

ENSCA European Natu ral Sausage Casings Association.

EPA Agencia de Protección Ambiental de Estados Unidos .

FAO Organización de las Naciones Unidas para la Alimentación y la Agricultura.

FEDECARNE Federación Madri leña de Detallistas de la Carne.

HHS United States Department of Health and Human Services .

IARC Agencia Internacional para la Investigación del Cáncer.

INE In stituto Nacional de Estadística.

INSCA International Na tural Sausage Casing Association.

JECFA Comité Mixto FAO/OMS de Expertos en Aditivos Alimentarios .

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MAGRAMA Ministerio de Agricultura , Alimentación y Medio Ambiente.

OMS Organización Mundial de la Salud .

SCF Scientific Committee on Food of European Com mission.

SCTs Servicios Científico Técnicos de la Universidad de Oviedo.

UE Unión Europea.

USA United States of America.

USDA United States Department of Agriculture .

WCRF World Cancer Research F und.

8.3.2 ABREVIATURAS

BaP Benzo(a)pireno.

BaP d12 Patrón interno Benzo(a)pireno deuterado.

CCR Cáncer colorectal.

ChN Chorizo embutido en tripa natural.

Ch S Chorizo embutido en tripa sintética.

GC Cromatógrafo de gases

HAP Hidrocarburos aromáticos policíclicos

HAP 4 Suma de los siguientes 4 h idrocarburos aromáticos policíclicos : benzo(a)pireno, benzo(a)antraceno, benzo(b)fluoranteno y criseno .

IGP Indicación Geográfica Protegida.

LOD Límite de detección.

LOQ Límite de cuantificación.

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MS Espectrómetro de masas.

O. E Objetivo específico.

O.G Ob jetivo general.

PVDC Cloruro de polivinilideno.

SCAN Full scan spectrum analysis .

SEM Microscop ía electrónica de barrido.

SIM Selected Ion Monitoring (Monitorización selectiva de iones).

S/N Relación señal/ruido.

8.3.3 SÍMBOLOS

a* Parámetro del espacio de color CIELAB. Representa la variación entre el magenta y el verde (valores negativos indican verde mientras valores positivos indican magenta).

b* Parámetro del espacio de color CIELAB. Representa la variación entre el amarillo y el azul (valores negativos indican azul y valores positivos indican amarillo).

BI Indice de pardeamiento ó enmarronecimiento .

d Distancia (m).

Hr Humedad relativa (%).

L* Parámetro del espacio de color CIELAB. Representa la luminosidad de color (L*= 0 indica negro y L* = 100 indica blanco).

T Temperatura (ºC).

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8.3.4 SIMBOLOS CON LETRAS GRIEGAS

•E Cambio total de color.

µL Microlitros.

Densidad de la mezcla. M

Densidad del hexano a 20 ºC (0,660 g/mL). hexanohex

Densidad del diclorometano a 20 ºC (1,325 g/mL). dicdiclorome tan o

Fracción en peso del hexano. x hexano

Fracción en peso del diclorometano. x diclorome tan o

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